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Abstract:

Conjugates of hydroxypropyl methacrylamide (HPMA)-derived copolymers
having attached thereto TNP-470 and a high load (e.g., higher than 3 mol
%) of alendronate (ALN), and processes of preparing same are disclosed.
Conjugates of polymers or copolymers having attached thereto an
anti-angiogenesis agent and an oligoaspartate bone targeting agent, and
processes of preparing same, are further disclosed.
Pharmaceutical compositions containing these conjugates and uses thereof
in the treatment and monitoring of bone related disorders are also
disclosed.

Claims:

1-60. (canceled)

61. A polymeric conjugate comprising an
(N-(2-hydroxypropyl)methacrylamide)polymeric backbone having attached
thereto TNP-470 and alendronate, wherein a load of said alendronate in
the polymeric conjugate is greater than 3 mol %.

62. The polymeric conjugate of claim 61, wherein said polymeric backbone
comprises a plurality of methacrylamide backbone units, whereas said
TNP-470 is attached to a portion of said backbone units and said
alendronate is attached to another portion of said backbone units.

63. The conjugate of claim 61, wherein said load of said alendronate is
greater than 5 mol %.

64. The conjugate of claim 63, wherein said load of said alendronate is
about 7 mol %.

65. The conjugate of claim 61, wherein at least one of said TNP-470 and
said alendronate is attached to said polymeric backbone via a linker.

66. The conjugate of claim 65, wherein said linker is a biodegradable
linker.

67. The conjugate of claim 66, wherein said biodegradable linker is an
enzymatically cleavable linker.

68. The conjugate of claim 67, wherein said enzymatically cleavable
linker is cleaved by Cathepsin K.

69. The conjugate of claim 68, wherein said linker comprises a
-[Gly-Gly-Pro-Nle]- oligopeptide.

70. The conjugate of claim 61, having the general formula II:
##STR00008## wherein: B is said TNP-470; D is said alendronate; each of
said L1 and L2 is independently said linker; x is an integer
having a value such that x/(x+y+w) multiplied by 100 is in the range of
from 0.01 to 99.9; y is an integer having a value such that y/(x+y+w)
multiplied by 100 is in the range of from 0.01 to 99.9; and w is an
integer having a value such that w/(x+y+w) multiplied by 100 is in the
range of from 3 to 99.9.

71. The conjugate of claim 61, having the structure: ##STR00009##
wherein: x is an integer having a value such that x/(x+y+w) multiplied by
100 is in the range of from 0.01 to 99.9; y is an integer having a value
such that y/(x+y+w) multiplied by 100 is in the range of from 0.01 to
99.9; and w is an integer having a value such that w/(x+y+w) multiplied
by 100 is in the range of from 3 to 99.9.

72. The conjugate of claim 70, wherein: x is an integer having a value
such that x/(x+y+w) multiplied by 100 is in the range of from 70 to 99.9;
y is an integer having a value such that y/(x+y+w) multiplied by 100 is
in the range of from 0.01 to 15; and w is an integer having a value such
that w/(x+y+w) multiplied by 100 is in the range of from 5 to 20.

73. The conjugate of claim 61, further comprising a labeling agent
attached thereto.

74. The conjugate of claim 73, having the structure: ##STR00010##
wherein: x is an integer having a value such that x/(x+y+w+z) multiplied
by 100 is in the range from 0.01 to 99.9; y is an integer having a value
such that y/(x+y+w+z) multiplied by 100 is in the range of from 0.01 to
99.9; w is an integer having a value such that w/(x+y+w+z) multiplied by
100 is in the range of from 3 to 99; and z is an integer having a value
such that x/(x+y+w+z) multiplied by 100 is in the range of from 0.01 to
99.9.

75. The conjugate of claim 61, having a polydispersity index ranging from
1 to 1.4.

76. The conjugate of claim 61, having a mean size distribution lower than
150 nm.

77. A pharmaceutical composition comprising, as an active ingredient, the
conjugate of claim 61 and a pharmaceutically acceptable carrier.

78. The pharmaceutical composition of claim 77, being packaged in a
packaging material and identified in print, in or on said packaging
material, for use in the treatment of, or in monitoring, a bone related
disease or disorder.

79. The pharmaceutical composition of claim 78, wherein said disease or
disorder is associated with angiogenesis.

80. The pharmaceutical composition of claim 79, wherein said disease or
disorder is selected from the group consisting of bone metastases and
bone cancer.

81. A method of treating a bone related disease or disorder in a subject
in need thereof, the method comprising administering to the subject a
therapeutically effective amount of the conjugate of claim 61.

82. The method of claim 81, wherein said disease or disorder is
associated with angiogenesis.

83. The method of claim 82, wherein said disease or disorder is selected
from the group consisting of bone metastases and bone cancer.

84. A method of monitoring a bone related disease or disorder in a
subject, the method comprising: administering to the subject the
conjugate of claim 73; and employing an imaging technique for monitoring
a distribution of the conjugate within the body or a portion thereof.

85. The method of claim 84, wherein said disease or disorder is selected
from the group consisting of bone metastases and bone cancer.

86. A process of synthesizing the conjugate of claim 61, the process
comprising: (a) coupling alendronate to N-(2-hydroxypropyl)methacrylamide
monomeric units, to thereby obtain alendronate-containing methacrylamide
monomeric units; (b) co-polymerizing N-(2-hydroxypropyl)methacrylamide
monomeric units, and/or ((N-(2-hydroxypropyl)methacrylamide) oligomeric
or polymeric units with said alendronate-containing methacrylamide
monomeric units and with methacrylamide monomeric units terminating with
a first reactive group, to thereby obtain a polymeric backbone which
comprises a plurality of methacrylamide backbone units in which a portion
of the backbone units has an alendronate attached thereto, and another
portion of the backbone units has said reactive group, said first
reactive group being capable of coupling TNP-470; and (c) coupling said
TNP-470 and said polymeric backbone via said first reactive group,
thereby obtaining the polymeric conjugate.

87. The process of claim 86, wherein said co-polymerizing is performed
via the Reversible addition-fragmentation chain transfer (RAFT)
technique.

88. A polymeric conjugate comprising a polymeric backbone having attached
thereto an anti-angiogenesis agent and a bone targeting moiety, said bone
targeting moiety being an oligopeptide which comprises from 2 to 100
aspartic acid residues.

89. The polymeric conjugate of claim 88, wherein said polymeric backbone
comprises a plurality of backbone units, and wherein a portion of said
plurality of backbone units has said anti-angiogenesis agent attached
thereto and another portion of said plurality of backbone units has said
oligopeptide of aspartic acid attached thereto.

92. The conjugate of claim 88, wherein said aspartic acid is D-aspartic
acid.

93. The conjugate of claim 88, wherein at least one of said
anti-angiogenesis agent and said oligopeptide is attached to said
polymeric backbone via a linker.

94. The conjugate of claim 88, wherein said polymeric backbone is derived
from a polymer selected from the group consisting of dextran, a water
soluble polyamino acid, a polyethylenglycol (PEG), a polyglutamic acid
(PGA), a polylactic acid (PLA), a polylactic-co-glycolic acid (PLGA), a
poly(D,L-lactide-co-glycolide) (PLA/PLGA), a
poly(hydroxyalkylmethacrylamide), a polyglycerol, a polyamidoamine
(PAMAM), and a polyethylenimine (PEI).

95. The conjugate of claim 88, further comprising a labeling agent
attached thereto.

96. The conjugate of claim 95, having the structure: ##STR00011##
wherein: a and b are each independently an integer having a value such
that a/(a+b+d) multiplied by 100 and/or b/(a+b+d)×100 are in the
range of from 0.01 to 15; and d is an integer having a value such that
d/(a+b+d) multiplied by 100 is in the range of from 70 to 99.9.

97. A pharmaceutical composition comprising, as an active ingredient, the
conjugate of claim 88 and a pharmaceutically acceptable carrier.

98. The pharmaceutical composition of claim 97, being packaged in a
packaging material and identified in print, in or on said packaging
material, for use in the treatment of, or in monitoring, a bone related
disease or disorder.

99. A method of treating a bone related disease or disorder in a subject
in need thereof, the method comprising administering to the subject a
therapeutically effective amount of the conjugate of claim 88.

100. A method of monitoring a bone related disease or disorder in a
subject, the method comprising: administering to the subject the
conjugate of claim 95; and employing an imaging technique for monitoring
a distribution of the conjugate within the body or a portion thereof.

101. A process of synthesizing the conjugate of claim 88, the process
comprising: (a) co-polymerizing a plurality of monomeric units of said
polymeric backbone, wherein a portion of said plurality comprises
monomeric units terminating by a first reactive group, and another
portion of said plurality comprises monomeric units terminating by a
second reactive group, to thereby obtain a co-polymer comprising a
polymeric backbone that comprises a plurality of backbone units, wherein
a portion of said backbone units has said first reactive group and
another portion of said backbone units has said second reactive group,
said first reactive group being capable of reacting with said
anti-angiogenesis agent and said second reactive being capable of
reacting with said bone targeting moiety; (b) coupling said bone
targeting moiety to said co-polymer via said first reactive group,
thereby obtaining a bone targeting moiety-containing copolymer; and (c)
coupling said anti-angiogenesis agent to said co-polymer via said second
reactive group, thereby obtaining the conjugate.

102. The process of claim 101, wherein said co-polymerizing is performed
via the Reversible addition-fragmentation chain transfer (RAFT)
technique.

Description:

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention, in some embodiments thereof, relates to
chemical conjugates and their use in therapy and diagnosis and, more
particularly, but not exclusively, to chemical conjugates of a polymer,
an anti-angiogenesis agent and a targeting moiety, which are useful, for
example, in the treatment and monitoring of bone related diseases and
disorders such as bone cancer and bone metastases.

[0003] Osteosarcoma is the most common type of primary bone cancer and
classified as a malignant mesenchymal neoplasm in which the tumor
directly produces defective osteoid (immature bone). It is a highly
vascular and extremely destructive malignancy that most commonly arises
in the metaphyseal ends of long bones. Over the past two decades,
multimodality treatment consisting of aggressive chemotherapy combined
with radical surgical resection, has been the mainstay of osteosarcoma
management, with achievable 5 year survival rates of 50% to 70% in
patients who do not have metastatic disease at presentation. Several
strategies were proposed, such as immune-based therapy, tumor-suppressor
or suicide gene therapy, or anticancer drugs that are not commonly used
in osteosarcoma [Quan et al. Cancer Metastasis Rev 2006; 10: 707-713].
However, still one-third of patients die from this devastating cancer,
and for those with unresectable disease there are no curative systemic
therapies.

[0004] Prostate cancer is the most common cancer of males in
industrialized countries and the second leading cause of male cancer
mortality. Mortality in these patients is not due to primary tumor
growth, but rather due to complications caused by metastases to vital
organs. Prostate cancer predominantly metastasizes to bone, but other
organ sites are affected including the lung, liver, and adrenal gland.

[0005] Breast cancer also often metastasizes to bones.

[0006] Bone metastases incidence in patients with advanced metastatic
disease is approximately 70%. Bone metastases are associated with
considerable skeletal morbidity, including severe bone pain, pathologic
fracture, spinal cord or nerve root compressions, and hypercalcemia of
malignancy. Chemotherapy agents, hormonal deprivation and bisphosphonates
are the common treatments for advanced metastatic disease. However, with
time, the disease progresses to a phase when the standard therapy fails
to control the malignancy and further progresses to a highly
chemotherapy-resistant state.

[0010] The largest class of drugs that block angiogenesis are the
multi-targeted tyrosine kinase inhibitors (TKIs) that target the VEGF
receptor (VEGFR). These drugs such as sunitinib (Sutent, Pfizer),
Sorafenib (Nexavar, Bayer/Onyx Pharmaceuticals) and Erlotinib (Tarveca,
Gennentech/OSI/Roche) have the advantages of hitting multiple targets,
convenient oral administration, and cost effectiveness. While these drugs
exhibit promising efficacy, their use is limited by their lack of target
specificity, which leads to unexpected toxicity [Cabebe et al. Curr Treat
Options Oncol 2007; 8:15-27].

[0011] Novel targeted angiogenesis inhibitors, for use with or without
other anti-neoplastic agents have therefore been sought for. A major
impediment towards this effort has been the inability to determine
therapeutic efficacy, the lack of reliable surrogate markers of tumor
angiogenesis, and the complexity of interactions between multiple host
cells and malignant cells involved in tumor angiogenesis, which may limit
the use of a single anti-angiogenic agent. Another significant obstacle
is that the vast majority of clinically used anti-cancer and
anti-angiogenic drugs are small molecules that exhibit a short half-life
in the bloodstream and a high overall clearance rate. These low-molecular
weight drugs diffuse rapidly into healthy tissues and are distributed
evenly within the body. As a consequence, relatively small amounts of the
drug reach the target site, and therapy is associated with low efficacy
and severe side effects.

[0012] TNP-470 is a low molecular weight synthetic analogue of fumagillin,
which is capable of selectively inhibiting endothelial growth in vitro.
In clinical trials, this drug was found to slow tumor growth in many
patients with metastatic cancer and exhibited a promising efficacy when
used in combination with conventional chemotherapy. However, at the doses
required for tumor regression, many patients experienced neurotoxicity.
Due to its dose-limiting neurotoxicity, no further clinical studies were
conducted for using TNP-470 per se. It has been concluded that clinical
uses of TNP-470 should be performed with this agent being targeted to
tumor tissue, in order to increase its site specificity and reduce side
effects.

[0013] Water-soluble polymers such as N-(2-Hydroxypropyl)methacrylamide
copolymers (HPMA) are biocompatible, non-immunogenic and non-toxic
carriers that enable specific delivery into tumor tissue [Satchi-Fainaro
et al. Nat Med 2004; 10: 255-261]. These macromolecules do not diffuse
through normal blood vessels but rather accumulate selectively in the
tumor site because of the enhanced permeability and retention (EPR)
effect. This phenomenon of passive diffusion through the hyperpermeable
neovasculature and localization in the tumor interstitium is observed in
many solid tumors for macromolecular agents and lipids. Conjugation of
anti-cancer drugs such as TNP-470 with copolymers, such as HPMA, should
enable selective targeting of these drugs to tumor tissue and thus reduce
side effects. Furthermore, such copolymer-drug conjugates should restrict
the passage through the blood brain barrier and would prolong the
circulating half-life of the drugs, hence inhibiting the growth of tumor
endothelial and epithelia cells by exposing the cells to the conjugated
drugs in the circulation for a longer time compared to the free drugs.

[0014] An example of the favorable characteristics obtained by conjugation
of an anti-angiogenesis agent such as TNP-470 to HPMA has been described
by Satchi-Fainaro et al. in WO 03/086382. This patent application teaches
conjugates of water-soluble polymers and TNP-470, and their use as
anti-tumor agents, in particular their use as carriers of TNP-470 into
tumor vessels, and their effect on the neurotoxicity of TNP-470.
According to the teachings of WO 03/086382, an exemplary such conjugate,
HPMA-(TNP-470) conjugate (caplostatin), exhibited superior antitumor
activity together with a reduced level of toxicity, as compared with
TNP-470 alone. WO 03/086382 further suggests incorporation of a targeting
ligand, such as RGD or antibodies.

[0015] The use of HPMA-TNP-470 conjugate for the treatment of angiogenesis
related conditions has also been described in WO 03/086178.

[0016] Another example of the increased activity yet reduced toxicity
obtained by conjugation of anti-tumor drugs to water-soluble polymers is
presented in U.S. Pat. No. 6,884,817.

[0017] An HPMA copolymer conjugate of paclitaxel has been described by
Meerum Terwogt et al. [Anticancer drugs 2001; 12:315-323]. This conjugate
was aimed at improving drug solubility and providing controlled release
of paclitaxel. In this conjugate, the paclitaxel is linked to the HPMA
copolymer through an ester bond, and is hence released from the polymer
by non-tissue specific hydrolytic or enzymatic (esterases) degradation of
the ester bond, thereby inducing the commonly observed toxicities of
paclitaxel.

[0018] Bisphosphonates (BPs) such as alendronate are compounds with a
chemical structure similar to that of inorganic pyrophosphate (PPi), an
endogenous regulator of bone mineralization. Several bisphosphonates are
established as effective treatments in clinical disorders such as
osteoporosis, Paget's disease of bone, myeloma, and bone metastases.
Bisphosphonates, such as zoledronic acid, have been shown to inhibit
angiogenesis [Wood et al. J Pharmacol Exp Ther 2002; 302: 1055-1061]. The
pharmacokinetic profile of bisphosphonates, which exhibit a strong
affinity to bone mineral under physiological conditions, their low
toxicity and anti-angiogenic activity are advantageous for targeting to
tumors confined to bony tissues.

[0021] WO 2004/062588 teaches water soluble polymeric conjugates for bone
targeted drug delivery with improved pharmacokinetics parameters and
better water solubility of the loaded drugs. The polymeric drug delivery
systems taught by this application are based on hydroxypropyl
methacrylamide (HPMA) conjugates of bone-targeting drugs such as
alendronate and D-Asp8 together with a bone-related therapeutic
agent. The loading of alendronate and D-Asp8 (SEQ ID NO:1) onto the
HPMA copolymer was 0.494 mmol/gram and 0.762 mmol/gram respectively.

[0022] PK2 (FCE28069) is a HPMA copolymer-doxorubicin-galactosamine
conjugate, which was designed as a treatment for hepatocellular carcinoma
or secondary liver disease [Seymour et al. Journal of Clinical Oncology
2002; 20:1668-1676]. Doxorubicin is an anthracycline antibiotic with
limited solubility in physiological fluids, and is a well established
anti-neoplastic drug. Galactosamine binds to the hepatic
asialoglycoprotein receptor (ASGPR) thus serving as a specific hepatic
targeting moiety. These components are linked to the HPMA polymer via an
enzymatically biodegradable linker which permits the release of free
doxorubicin within the liver, thus increasing the drug concentration in
its site of action. The enzymatic degradable linker is a tetrapeptide
spacer (Gly-Phe-Leu-Gly) (SEQ ID NO:2), designed for cleavage by
lysosomal cathepsins.

[0025] Currently known agents used for treating bone related cancer and
other angiogenesis-related conditions, at doses where anti-tumor activity
is achieved, are characterized by high toxicity, which limits their use.
In a search for modes of modifying currently known anti-angiogenesis
agents so as to enable higher therapeutic efficacy thereof together with
a reduced level of side effects, the present inventors have designed and
successfully prepared and practiced a novel polymeric conjugate of
N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer, TNP-470 and the bone
targeting agent alendronate (ALN), wherein the TNP-470 and alendronate
molecules are conjugated to backbone units of the HPMA polymeric backbone
via biodegradable linkers and the percent of alendronate loaded onto the
HPMA polymeric backbone is higher than in currently known
alendronate-polymer conjugates (e.g., is greater than 3 mol % of the
polymeric conjugate).

[0026] The present inventors have further devised and successfully
practiced a novel process for preparing the conjugates described herein,
while obtaining a high load of alendronate in the polymer as well as a
homogenous size distribution, i.e. low polydispersity, of the polymer.
This process can be beneficially performed in a controlled manner at
30° C.

[0027] The present inventors have designed and successfully prepared and
practiced a novel conjugate of a polymer (e.g., a
N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer), an anti-angiogenesis
agent (e.g., TNP-470) and a bone targeting agent being D-Asp8 (SEQ ID
NO:1).

[0028] According to an aspect of some embodiments of the present invention
there is provided a conjugate comprising an
N-(2-hydroxypropyl)methacrylamide)-derived polymeric backbone having
attached thereto TNP-470 and alendronate, wherein a load of the
alendronate in the polymer is greater than 3 mol %

[0029] According to some embodiments of the invention, the load of the
alendronate in the polymer is greater than 5 mol %.

[0030] According to some embodiments of the invention, the load of the
alendronate in the polymer is about 7 mol %.

[0031] According to some embodiments of the invention, at least one of the
TNP-470 and the alendronate is attached to the polymer via a linker.

[0032] According to some embodiments of the invention, each of the TNP-470
and the alendronate is attached to the polymer via a linker.

[0033] According to some embodiments of the invention, the linker is a
biodegradable linker.

[0034] According to some embodiments of the invention, the biodegradable
linker is selected from the group consisting of a pH-sensitive linker and
an enzymatically cleavable linker.

[0035] According to some embodiments of the invention, the biodegradable
linker is an enzymatically cleavable linker.

[0036] According to some embodiments of the invention, the enzymatically
cleavable linker is cleaved by an enzyme which is expressed in tumor
tissues.

[0037] According to some embodiments of the invention, the enzymatically
cleavable linker is cleaved by an enzyme which is overexpressed in tumor
tissues.

[0038] According to some embodiments of the invention, the enzyme is
selected from a group consisting of Cathepsin B, Cathepsin K, Cathepsin
D, Cathepsin H, Cathepsin L, legumain, MMP-2 and MMP-9.

[0039] According to some embodiments of the invention, the enzyme is
Cathepsin K.

[0040] According to some embodiments of the invention, the linker
comprises an oligopeptide group containing from 2 to 10 amino acid
residues.

[0041] According to some embodiments of the invention, the oligopeptide is
-[Gly-Gly-Pro-Nle]- (SEQ ID NO:3).

[0042] According to some embodiments of the invention, the TNP-470 is
linked to the polymer or to the linker via a spacer.

[0043] According to some embodiments of the invention, the spacer has the
formula G-(CH2)n-K, wherein n is an integer from 1 to 4; and G and K
are each independently selected from the group consisting of NH, O and S.

[0044] According to some embodiments of the invention, G and K are each NH
and n is 2.

[0045] According to some embodiments of the invention, the alendronate is
attached to the polymer via a linker that comprises (SEQ ID NO:3).

[0046] According to some embodiments of the invention, the conjugate has
the general formula II, as described herein.

[0047] According to some embodiments of the invention, the conjugate has
the structure:

##STR00001##

[0048] According to some embodiments of the invention, x is an integer
that equals 70-99.9 and y and w are each independently an integer that
equals 0.01-15.

[0049] According to some embodiments of the invention, the conjugate
further comprising a labeling agent.

[0050] According to some embodiments of the invention, the labeling agent
is selected from the group consisting of a fluorescent agent, a
radioactive agent, a magnetic agent, a chromophore, a bioluminescent
agent, a chemiluminescent agent, a phosphorescent agent and a heavy metal
cluster.

[0051] According to some embodiments of the invention, the labeling agent
is Fluorescein isothiocyanate.

[0052] Such conjugate is having, for example, the following structure:

##STR00002##

[0053] wherein:

[0054] x is an integer that equals 70-99.9; and

[0055] y, z and w are each independently an integer that equals 0.01-15.

[0056] According to an aspect of some embodiments of the present invention
there is provided a process of synthesizing the conjugates as described
hereinabove, the process comprising:

[0058] (b) co-polymerizing N-(2-hydroxypropyl)methacrylamide monomeric
units, and/or ((N-(2-hydroxypropyl)methacrylamide) oligomeric or
polymeric units with the alendronate-containing methacrylamide monomeric
units and with methacrylamide monomeric units terminating with a first
reactive group, to thereby obtain a polymeric backbone which comprises a
plurality of methacrylamide backbone units in which a portion of the
backbone units has an alendronate attached thereto, and another portion
of the backbone units has said reactive group, the first reactive group
being capable of coupling TNP-470; and

[0059] (c) coupling the TNP-470 and the polymeric backbone via the first
reactive group, thereby obtaining the polymeric conjugate.

[0060] According to another aspect of some embodiments of the present
invention there is provided a conjugate comprising a polymeric backbone
having attached thereto an anti-angiogenesis agent and a bone targeting
moiety, the bone targeting moiety being an oligopeptide of aspartic acid
which comprises from 2 to 100 amino acid residues.

[0061] According to some embodiments of the invention, the oligopeptide
comprises from 2 to 20 amino acid residues.

[0062] According to some embodiments of the invention, the oligopeptide
comprises 8 amino acid residues.

[0063] According to some embodiments of the invention, the aspartic acid
is selected from the group consisting of D-aspartic acid and L-aspartic
acid.

[0064] According to some embodiments of the invention, the aspartic acid
is D-aspartic acid.

[0065] According to some embodiments of the invention, at least one of the
anti-angiogenesis agent and the bone targeting moiety is attached to the
polymeric backbone via a linker.

[0066] According to some embodiments of the invention, the linker is a
biodegradable linker.

[0067] According to some embodiments of the invention, each of the
anti-angiogenesis agent and the bone targeting moiety is attached to the
polymeric backbone via a linker:

[0068] According to some embodiments of the invention, the polymeric
backbone is derived from a polymer that has an average molecular weight
that ranges from 100 Da to 800 kDa.

[0069] According to some embodiments of the invention, the polymeric
backbone is derived from a polymer selected from the group consisting of
dextran, a water soluble polyamino acid, a polyethylenglycol (PEG), a
polyglutamic acid (PGA), a polylactic acid (PLA), a
polylactic-co-glycolic acid (PLGA), a poly(D,L-lactide-co-glycolide)
(PLA/PLGA), a poly(hydroxyalkylmethacrylamide), a polyglycerol, a
polyamidoamine (PAMAM), and a polyethylenimine (PEI).

[0070] According to some embodiments of the invention, the polymer is
N-(2-hydroxypropyl)methacrylamide).

[0071] According to some embodiments of the invention, the
anti-angiogenesis agent is TNP-470.

[0072] According to some embodiments of the invention, the biodegradable
linker is selected from the group consisting of a pH-sensitive linker and
an enzymatically-cleavable linker, as described herein.

[0073] According to an aspect of some embodiments of the present invention
there is provided a pharmaceutical composition comprising, as an active
ingredient, any of the conjugates described herein and a pharmaceutically
acceptable carrier.

[0074] According to some embodiments of the invention, the composition is
packaged in a packaging material and identified in print, in or on the
packaging material, for use in the treatment of a bone related disease or
disorder.

[0075] According to some embodiments of the invention, the conjugate
comprises a labeling agent, the composition being packaged in a packaging
material and identified in print, in or on the packaging material, for
use in monitoring a bone related disease or disorder.

[0076] According to some embodiments of the invention, the bone related
disease or disorder is associated with angiogenesis.

[0077] According to an aspect of some embodiments of the present invention
there is provided a method of treating a bone related disease or disorder
in a subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of any of the conjugates
described herein.

[0078] According to an aspect of some embodiments of the present invention
there is provided a use of the conjugate as described hereinabove as a
medicament.

[0079] According to an aspect of some embodiments of the present invention
there is provided a use of the conjugate as described hereinabove in the
manufacture of a medicament for treating a bone-related disease or
disorder.

[0080] Unless otherwise defined, all technical and/or scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
methods and materials similar or equivalent to those described herein can
be used in the practice or testing of embodiments of the invention,
exemplary methods and/or materials are described below. In case of
conflict, the patent specification, including definitions, will control.
In addition, the materials, methods, and examples are illustrative only
and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Some embodiments of the invention are herein described, by way of
example only, with reference to the accompanying drawings and images.
With specific reference now to the drawings in detail, it is stressed
that the particulars shown are by way of example and for purposes of
illustrative discussion of embodiments of the invention. In this regard,
the description taken with the drawings makes apparent to those skilled
in the art how embodiments of the invention may be practiced.

[0082] In the drawings:

[0083] FIG. 1 presents a scheme illustrating the synthesis of an HPMA
copolymer-ALN-TNP-470 conjugate according to some embodiments of the
present invention.

[0085] FIGS. 3A-G present the 2-D chemical structure of
fluorosceinated-HPMA copolymer-ALN-TNP-470 conjugates according to some
embodiments of the present invention (FIG. 3A); a diagram showing FPLC
detection of unbound HPMA-ALN-TNP470 conjugate in the samples, in the
presence and absence of Hydroxyapitate, at selected time points (FIG.
3B); and plots showing the percentages of HPMA-ALN-TNP-470 conjugate
bound to hydroxyapatite as a function of the elution time (FIG. 3C); size
exclusion chromatography (SEC) profile of conjugate polymerized by the
classical polymerization method (Polymerization I conjugate; FIG. 3D) or
conjugate polymerized by RAFT polymerization (Polymerization II
conjugate; FIG. 3E); a graph showing the hydrodynamic diameter size
distribution of the HPMA-ALN-TNP-470 polymerization I conjugate and
polymerization II conjugate (FIG. 3F); and an image of the polymer I
conjugate particles obtained (FIG. 3G).

[0087] FIG. 5 present comparative plots demonstrating that ALN and TNP-470
retain their antiangiogenic effect when bound to the HPMA copolymer. The
percentage of average cell growth is similar in the presence of
polymer-conjugated ALN and TNP-470 in HUVEC cells (closed triangles),
Saos-2 cells (closed squares) and MG-63-Ras (closed diamonds) compared
with a combination of free ALN and free TNP-470 in HUVEC cells (open
triangles), in Saos-2 cells (open squares) and MAG-63-Ras (open diamonds.
The inhibition of endothelial proliferation by the conjugate was reduced
significantly (IC50=4200 nM) in the presence of cathepsin K
inhibitor III (open circles). Solid and dashed lines represent the
proliferation of cells in the presence (solid line) or absence (dashed
line) of growth factors. Data represent mean±SD.

[0090] FIG. 8 presents a scheme illustrating the synthesis of two HPMA
copolymer-D-Asp8-TNP-470 conjugates (HP1 and HP2) (D-Asp8
having SEQ ID NO:1) according to some embodiments of the present
invention.

[0095] The present invention, in some embodiments thereof, relates to
chemical conjugates and their use in therapy and diagnosis and, more
particularly, but not exclusively, to chemical conjugates of a polymer,
an anti-angiogenesis agent and a targeting moiety, which are useful, for
example, in the treatment and monitoring of bone related diseases and
disorders such as bone cancer and bone metastases.

[0096] The principles and operation of the conjugates, compositions, use,
methods and processes according to the invention may be better understood
with reference to the drawings and accompanying descriptions.

[0097] Before explaining at least one embodiment of the invention in
detail, it is to be understood that the invention is not limited in its
application to the details set forth in the following description or
exemplified by the Examples. The invention is capable of other
embodiments or of being practiced or carried out in various ways. Also,
it is to be understood that the phraseology and terminology employed
herein is for the purpose of description and should not be regarded as
limiting.

[0098] As discussed hereinabove, currently known agents used for treating
bone related cancer and other angiogenesis-related conditions, at doses
where anti-tumor activity is achieved, are characterized by high
toxicity, which limits their use.

[0099] The present inventors have now devised and successfully prepared
and practiced novel conjugates of a copolymer having attached thereto an
anti-angiogenesis agent and a bone targeting moiety.

[0100] More specifically, but not exclusively, the present inventors have
devised and successfully practiced novel processes of preparing such
conjugates, in which the bone targeting moiety is alendronate or an
oligoaspartate.

[0101] The present inventors have devised and successfully practiced novel
processes of preparing such conjugates in which alendronate is present in
a relatively high load within the copolymer.

[0102] The present inventors have surprisingly uncovered that alendronate
and TNP-470 can act in synergy in inhibition of angiogenesis. Taken
together with the dose-dependent anti-angiogenesis activity of
alendronate, the alendronate high-loaded conjugates described herein are
therefore characterized as highly potent agents for treating bone-related
diseases and disorders.

[0103] As demonstrated in the Examples section that follows, the present
inventors have successfully prepared and practiced a novel polymeric
conjugate of a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, having
attached thereto TNP-470 and the bone targeting agent, alendronate (ALN),
wherein the TNP-470 and alendronate are conjugated to backbone units of
the HPMA-derived polymeric backbone via biodegradable linkers and the mol
percent of alendronate loaded onto the conjugate is higher than in
currently known alendronate-polymer conjugates (e.g., is greater than 3
mol % of the polymeric conjugate).

[0105] The present inventors have further designed and successfully
prepared and practiced a novel conjugate of a polymer (e.g., a
N-(2-hydroxypropyl)methacrylamide (HPMA)-derived co-polymer) having
attached thereto an anti-angiogenesis agent (e.g., TNP-470) and a bone
targeting agent being D-Asp8 (SEQ ID NO:1).

[0106] Thus, according to one aspect of some embodiments of the invention
there is provided a polymeric conjugate comprising an
N-(2-hydroxypropyl)methacrylamide)-derived polymeric backbone having
attached thereto TNP-470 and alendronate, wherein a load of the
alendronate in the polymeric conjugate is greater than 3 mol %.

[0107] N-(2-hydroxypropyl)methacrylamide (HPMA) polymers are a class of
water-soluble synthetic polymeric carriers that have been extensively
characterized as biocompatible, non-immunogenic and non-toxic. HPMA
polymers can be tailored through relatively simple chemical
modifications, in order to regulate their respective drug and targeting
moiety content. Further, the molecular weight and charge of these
polymers may be manipulated so as to allow renal clearance and excretion
from the body, or to alter biodistribution while allowing tumor
targeting.

[0108] The tumor targeting capacity of HPMA polymers is attributed, at
least in part, to the enhanced permeability and retention (EPR) effect of
such polymers. Thus, HPMA conjugates are characterized by a limited
diffusion and/or extravasation through normal blood vessels, due to the
high molecular weight and hydrodynamic diameter thereof, and therefore
accumulate selectively at the tumor site, which is characterized by leaky
blood vessels having abnormal form and architecture and wide
fenestrations, pores and vesicular vacuolar organelles (VVO). Due to the
poor lymphatic drainage from tumor, macromolecules tend to be retained in
the tumor microenvironment. Conjugating drugs to polymers such as HPMA is
also expected to restrict the passage of the conjugate through the blood
brain barrier, thus prolonging the circulating half-life of the drugs and
abrogating neurotoxicity associated with many chemotherapeutic and
anti-angiogenic drugs.

[0109] A polymeric conjugate, comprising an
N-(2-hydroxypropyl)methacrylamide)-derived polymeric backbone having
attached thereto TNP-470 and alendronate, that has a molecular weight
higher than 10 kDa typically exhibit an EPR effect, as described herein,
while polymeric substances that have a molecular weight of 100 kDa and
higher have relatively long half-lives in plasma and an inefficient renal
clearance. Accordingly, a molecular weight of the polymeric conjugate can
be determined while considering the half-life in plasma, the renal
clearance, and the accumulation in the tumor of the conjugate. The
molecular weight of the polymeric conjugate can be controlled, at least
to some extent, by the degree of polymerization (or co-polymerization).

[0110] In some embodiments, the conjugate described herein has a MW that
ranges from 100 Daltons to 800 kDa. In some embodiments, the conjugate
described herein has a MW that ranges from 10 kDa to 800 kDa. In some
embodiments, the conjugate has a MW that ranges from 10 kDa to 60 kDa.

[0111] In some embodiments of the invention, the conjugates described
herein comprise an HPMA polymeric backbone comprised of
N-(2-hydroxypropyl)methacrylamide-derived backbone units (a polymeric
backbone formed by polymerizing N-(2-hydroxypropyl)methacrylamide
monomers), whereby TNP-470 molecules are attached to a portion of these
backbone units and alendronate molecules are attached to another portion
of these backbone units, as described herein. Those backbone units within
the polymeric backbone that are not linked to another moiety (e.g.,
TNP-470, alendronate, or any of the other moieties described herein) are
referred to herein as "free" or "non-functionalized" backbone units.

[0112] Since the polymeric backbone in the conjugates described herein is
composed of some backbone units that have alendronate attached thereto,
some backbone units that have TNP-470 attached thereto, and optionally
some free backbone units, these conjugates represent HPMA-derived
co-polymers.

[0113] As discussed hereinabove, TNP-470 is a potent anti-angiogenesis
agent. Its use as a free drug has been limited by its low solubility and
dose-dependent neurotoxicity.

[0114] The phrase "anti-angiogenesis agent", which is also referred to
herein interchangeably as "anti-angiogenic agent" or "angiogenesis
inhibitor", describes an agent having the ability to (a) inhibit
endothelial cell proliferation or migration; (b) kill proliferating
endothelial cells; and/or (c) inhibit the formation of new blood vessels
in a tissue.

[0115] As further discussed hereinabove, alendronate
(4-amino-1-hydroxybutylidene) bisphosphonic acid) is a bisphosphonate
which exhibits a strong affinity to bone minerals under physiological
conditions.

[0117] It has been previously shown that alendronate exhibits
anti-angiogenesis activity in a dose dependent manner. For example, Cheng
et al. have shown that alendronate reduces the mRNA level and cellular
level of Matrix metalloproteinase-2 (MMP-2) enzyme in osteosarcoma cell
lines in a time and dose-dependent manner [Cheng et al. 2004, Pediatr
Blood Cancer 42; 410-415].

[0118] As demonstrated in the Examples section that follows, the
anti-angiogenesis activity of alendronate is dose dependent, as assessed
by the extent of HUVEC and Saos-2 human osteosarcoma cell line
proliferation inhibition, whereby the extent of inhibition is
proportional to the alendronate concentrations, i.e., at higher
alendronate concentration a stronger anti-angiogenesis activity could be
observed.

[0119] Conjugating alendronate and an anti-angiogenesis agent to polymers
is highly beneficial for producing an agent that is characterized by both
selectivity, due to the EPR effect attributed to the polymer, and the
bone-targeting effect attributed to the alendronate, and a potent
therapeutic activity, due to the presence of a potent anti-angiogenesis
agent.

[0120] However, such conjugates, which are further characterized by a high
load of alendronate, as described herein, are even more potent, due to
the dual targeting and anti-angiogenesis effect that can be potentially
exhibited by the alendronate.

[0121] According to some embodiments of the invention, the conjugates
described herein are characterized by an alendronate loading which is
higher than 3 mol %.

[0122] Herein, the phrase "loading", or simply "load", and any grammatical
diversion thereof, is used to describe the amount of an agent that is
attached to the polymeric backbone of the conjugates described herein,
and is represented herein by the mol % of this agent in the conjugate, as
defined hereinafter.

[0123] As used herein, the term "mol %" describes the number of moles of
an attached moiety per 1 mol of the polymeric conjugate, multiplied by
100.

[0130] As demonstrated in the Examples section that follows, at such high
loads, alendronate can exhibit both a bone targeting effect and an
anti-angiogenesis effect.

[0131] As further demonstrated in the Examples section that follows, it
has been further surprisingly uncovered that a combined therapy of
TNP-470 and alendronate results in a synergistic anti-angiogenic
activity, when alendronate is utilized at a high concentration.

[0132] As demonstrated in the Examples section that follows, the
anti-angiogenesis activity of alendronate and TNP-470, when administered
together, as demonstrated by their inhibitory effect on the proliferation
of endothelial cells, was superior to the cumulative anti-angiogenesis
activity of each agent when administered alone (see, FIG. 2). This
synergistic activity could be observed only at high alendronate
concentration whereas at low alendronate concentration (lower than 100
nM), no synergistic activity could be detected. These results show that
the synergistic activity between alendronate and TNP-470 is
dose-dependent. As further demonstrated in the Examples section that
follows, the in vivo inhibition of osteosarcoma tumor growth in mice was
significantly enhanced when conjugating both TNP-470 and alendronate to
HPMA, with a high load of alendronate (see, FIG. 7D), as compared to the
inhibition observed when equivalent concentration of free alendronate and
free TNP-470 were administered (i.e., unconjugated). It can therefore be
clearly deduced that the synergistic anti-angiogenesis activity results
from the relatively high concentration (load) of alendronate within the
conjugate.

[0133] Since alendronate exhibits a dose-dependent anti-angiogenesis
activity, and further since it is shown herein that alendronate and
TNP-470 can act in synergy in inhibition of angiogenesis, such
high-loaded conjugates can be beneficially utilized in the treatment of
bone-related diseases and disorders such as those conditions that are
associated with angiogenesis.

[0134] Thus, in some embodiments of the invention, the TNP-470 and
alendronate that are attached to the polymer in the conjugates described
herein act in synergy.

[0135] The phrase "synergy" or "synergistic activity", as used herein and
in the art, describes a cooperative action encountered in combinations of
two or more biologically active agents in which the combined effect
exhibited by the two agents when used together exceeds the sum of the
effect of each of the agents when used alone.

[0136] "Synergy" or "synergistic activity" is therefore often determined
when a value representing an effect of a combination of two active agents
is greater than the sum of the same values obtained for each of these
agents when acting alone.

[0137] A synergy between two anti-angiogenesis agents may be determined by
methods well known in the art.

[0138] In each of the conjugates described herein, the alendronate and the
TNP-470 can each be linked to the polymeric backbone directly, or
indirectly, through a linker moiety (also referred to herein as a linker,
a linker group or a linking group), whereby, in some embodiments, the
direct/indirect linkage is designed as being cleavable at conditions
characterizing the environment of a desired bodily site (e.g., by certain
enzymes or pH), as detailed hereinbelow.

[0139] Hence, according to some embodiments of the invention, at least one
of the TNP-470 and the alendronate is attached to the polymeric backbone
via a linker. In some embodiments, each of the TNP-470 and the
alendronate is attached to the polymeric backbone via a linker. The
linker linking the TNP-470 to the polymer and the linker linking the
alendronate to the polymeric backbone may be the same or different.

[0140] The linker described herein refers to a chemical moiety that serves
to couple the TNP-470 and/or the alendronate to the polymeric backbone
while not adversely affecting either the targeting function of the
alendronate or the therapeutic effect of the alendronate and/or the
TNP-470.

[0141] In some embodiments, the linker is a biodegradable linker.

[0142] The phrase "biodegradable linker", as used herein, describes a
linker that is capable of being degraded, or cleaved, when exposed to
physiological conditions. Such physiological conditions can be, for
example, pH, a certain enzyme, and the like.

[0143] In some embodiments, the linker is capable of being cleaved by
pre-selected cellular enzymes, for instance, those found in osteoblasts,
osteoclasts, lysosomes of cancerous cells or proliferating endothelial
cells. Alternatively, an acid hydrolysable linker could comprise an ester
or amide linkage and be for instance, a cis-aconityl linkage. Such
linkers further enhance the therapeutic activity and reduced toxicity of
the conjugates described herein, by allowing the release of the
anti-angiogenesis drug and/or the alendronate only at the desired bodily
site.

[0144] Accordingly, according to some embodiments, the biodegradable
linker is a pH-sensitive linker or an enzymatically-cleavable linker.

[0145] A pH-sensitive linker comprises a chemical moiety that is cleaved
or degraded only when subjected to a certain pH condition, such as acidic
pH (e.g., lower than 7), neutral pH (6.5-7) or basic pH (higher than 7).

[0146] Such a linker may, for example, be designed to undergo hydrolysis
under acidic or basic conditions, and thus, the conjugate remains intact
and does not release the agents attached to the polymer in the body,
until its reaches a physiological environment where a pH is either acidic
or basic, respectively.

[0148] In some embodiments, the biodegradable linker is an
enzymatically-cleavable linker. Such a linker is typically designed so as
to include a chemical moiety, typically, but not exclusively, an amino
acid sequence, that is recognized by a pre-selected enzyme. Such an amino
acid sequence is often referred to in the art as a "recognition motif". A
conjugate comprising such a linker typically remains substantially intact
in the absence of the pre-selected enzyme, and hence does not cleave or
degrade so as to the release the agent attached thereto until it reaches
an environment where this enzyme is present at a substantial
concentration.

[0149] In some embodiments, the enzymatically cleavable linker is cleaved
by an enzyme which is expressed in tumor tissues. A conjugate comprising
such a linker ensures, for example, that a substantial amount of the
conjugated TNP-470 is released from the conjugate only at the tumor
tissue, thus reducing the side effects associated with the non-selective
administration of the drug.

[0150] In some embodiments, the enzymatically cleavable linker is cleaved
by an enzyme which is overexpressed in tumor tissues.

[0151] Exemplary enzymes which are suitable for use in the context of
these embodiments include, but are not limited to, Cathepsin B, Cathepsin
K, Cathepsin D, Cathepsin H, Cathepsin L, legumain, MMP-2 and MMP-9.

[0152] Suitable linkers include, but are not limited to, alkyl chains;
alkyl chains optionally substituted with one or more substituents and in
which one or more carbon atoms are optionally interrupted by a nitrogen,
oxygen and/or sulfur heteroatom.

[0154] Such alkyl chains and/or oligopeptides can optionally be
functionalized so as allow their covalent binding to the moieties linked
thereby (e.g., the polymeric backbone and the alendronate, the polymeric
backbone and the TNP-470). Such a functionalization may include
incorporation or generation of reactive groups that participate in such
covalent bindings.

[0155] In some embodiments, the linker is a biodegradable oligopeptide
which contains, for example, from 2 to 10 amino acid residues.

[0156] In some embodiments, the linker is a Cathepsin K-cleavable linker.

[0160] Other Cathepsin K cleavable sites are those that include a
-[Gly-Pro-Nle]- moiety (SEQ ID NO: 5). Another example include
[-Gly-Gly-NH--C6-Gly-Pro-Nle]- (SEQ ID NO: 6).

[0161] As demonstrated in the Examples section that follows, a Cathepsin K
cleavable linker being -[Gly-Gly-Pro-Nle]- (SEQ ID NO:3) was used,
linking both alendronate and TNP-470 to the HPMA polymeric backbone (see,
FIG. 1). As further demonstrated in the Examples section that follows, a
HPMA copolymer-ALN-TNP-470 conjugate comprising such linker moieties
successfully inhibited proliferation of endothelial cells as well as
Saos-2 and MG-63-Ras human osteosarcoma cells. The involvement of
Cathepsin K in the release of the TNP-470 and alendronate from the
polymer could be deduced from the reduced activity of the conjugate when
incubated together with a cathepsin K inhibitor whereby the conjugate
inhibited the proliferation of HUVEC at a 4-logs higher concentration in
the presence of cathepsin K inhibitor III than in its absence (see, FIG.
5).

[0162] An oligopeptide linker which contains the pre-selected amino acid
sequence (recognition motif) can also be constructed such that the
recognition motif is repeated several times within the linker, to thereby
enhance the selective release of the attached agent. Various recognition
motifs of the same or different enzymes can also be incorporated within
the linker. Similarly, the linker may comprise multiple pH sensitive
bonds or moieties. Linkers comprising such multiple cleavable sites can
enhance the selective release of the anti-angiogenesis agent at the
desired bodily site, thereby reducing adverse side effects, and further
enhance the relative concentration of the released drug at the bodily
site when it exhibits its activity.

[0163] In cases where the TNP-470 and/or the alendronate is bound directly
to the polymeric backbone, the bond linking these moieties can also be
biodegradable, for example, an enzymatically-cleavable bond or a
pH-sensitive bond (e.g., an acid-hydrolyzable bond). Such a bond can be
formed upon functionalizing backbone units of the polymeric backbone, the
alendronate and/or the TNP-470, so as to include compatible reactive
groups for forming the desired bond.

[0164] In some embodiments, the TNP-470 is linked to the polymer or to the
linker via a spacer. In some embodiments, the alendronate is linked to
the polymer or to the linker via a spacer. The spacers can be the same or
different.

[0165] The term "spacer" as used herein describes a chemical moiety that
is covalently attached to, and interposed between, the polymeric backbone
and the linker, the TNP-470 and/or the alendronate, thereby forming a
bridge-like structure between the polymeric backbone and the linker, the
TNP-470 and/or the alendronate. In some embodiments, the spacer does not
actively participate in a biological process, and is present in the
conjugate for the purpose of facilitating its synthesis and/or improving
its performance in terms of, for example, steric considerations, as is
detailed hereinbelow.

[0166] Suitable spacers include, but are not limited to, alkylene chains,
optionally substituted by one or more substituents and which are
optionally interrupted by one or more nitrogen, oxygen and/or sulfur
heteroatom.

[0167] Other suitable spacers include amino acids and amino acid
sequences, optionally functionalized with one or more reactive groups for
being coupled to the polymeric backbone, TNP-470 and/or alendronate.

[0168] In some embodiments, the spacer has the formula G-(CH2)n-K,
wherein n is an integer from 1 to 10; and G and K are each a reactive
group, as defined herein, such as, for example, NH, O or S. In some
embodiments, G and K are each NH and n is 2.

[0169] In some embodiments, the spacer is an amino acid sequence,
optionally an inert amino acid sequence (namely, does not affect the
affinity or selectivity of the conjugate). Such a spacer can be utilized
for elongating or functionalizing the linker.

[0170] In some cases, a spacer is utilized for enabling a more efficient
and simpler attachment of the alendronate and/or the TNP-470 to the
polymeric backbone or linker, in terms of steric considerations (renders
the site of the polymeric backbone to which coupling is effected less
hindered) or chemical reactivity considerations (adds a compatible
reactive group to the site of the polymeric backbone to which coupling is
effected). In some cases, the spacer may contribute to the performance of
the resulting conjugate. For example, the spacer may render an
enzymatically cleavable linker less sterically hindered and hence more
susceptible to enzymatic interactions.

[0171] The spacer may also be used in order to attach other agents (e.g.,
a labeling agent, as described hereinbelow) to the conjugate.

[0172] The spacer may be varied in length and in composition, depending on
steric and chemical considerations, and may be used to space the TNP-470
and alendronate form the polymeric backbone and/or the linker.

[0173] As demonstrated in the Examples section that follows, the present
inventors have successfully synthesized a conjugate wherein the TNP-470
is linked to the HPMA polymeric backbone via a cathepsin K cleavable
linker being -[Gly-Gly-Pro-Nle]- (SEQ ID NO: 3) and a spacer being
--NH--(CH2)2--NH-- (see, FIG. 1).

[0174] As discussed hereinabove, the degree of loading of the TNP-470 and
alendronate may be expressed as mole %, as defined herein.

[0175] The optimal degree of loading of TNP-470 is determined empirically
based on the desired properties of the conjugate (e.g., water solubility,
therapeutic efficacy, pharmacokinetic properties, toxicity and dosage
requirements), and synthetic considerations (e.g., the amount of the drug
that can be attached to the backbone units in a certain synthetic
pathway).

[0176] In some embodiments, the loading of TNP-470 in the polymer is
greater than 1 mol %.

[0177] In some embodiments, the loading of the TNP-470 in the conjugate
ranges from 1 mol % to 90 mol %, from 1 mol % to 50 mol %, from 1 mol %
to 20 mol %, from 1 mol % to 10 mol %, or from 1 mol % to 5 mol %.

[0178] The number of backbone units in the polymeric backbone having
TNP-470 attached thereto is defined herein as "y", the number of backbone
units in the polymeric backbone having alendronate attached thereto is
herein defined as "w" and the number of free backbone units in the
polymeric backbone (which are not bound to an additional moiety) is
herein defined as "x".

[0179] Accordingly, in some embodiments, the conjugate described herein
can be represented by the general formula II:

##STR00003##

wherein:

[0180] x is an integer having a value such that x/(x+y+w) multiplied by
100 is in the range of from 0.01 to 99.9;

[0181] y is an integer having a value such that y/(x+y+w) multiplied by
100 is in the range of from 0.01 to 99.9; and

[0182] w is an integer having a value such that w/(x+y+w) multiplied by
100 is in the range of from 3 to 99; B is TNP-470;

[0183] D is alendronate; and

[0184] each of L1 and L2 is independently the linker, as
described herein.

[0185] In some embodiments, the conjugate has the following structure:

##STR00004##

[0186] wherein x, y and z are as defined herein.

[0187] According to some embodiments of the invention, x is an integer
having a value such that x/(x+y+w) multiplied by 100 is in the range of
from 70 to 99.9; y is an integer having a value such that y/(x+y+w)
multiplied by 100 is in the range of from 0.01 to 15; and w is an integer
having a value such that w/(x+y+w) multiplied by 100 is in the range of
from 5 to 20.

[0189] In some embodiments, the conjugate described herein further
comprises a labeling agent attached thereto.

[0190] In some embodiments, the labeling agent is attached to a portion of
the backbone units that do not have the TNP-470 or the alendronate
attached thereto.

[0191] In such cases the number of backbone units in the polymeric
backbone having the labeling agent attached thereto is defined as "z", as
shown in the general Formula hereinbelow.

[0192] The attachment of a labeling agent to the conjugate enables
utilizing these conjugates for monitoring bone related disease or
disorders, for example, monitoring the therapeutic effect exhibited by
the conjugate described herein, as well as its biodistribution.

[0193] As used herein, the phrase "labeling agent" describes a detectable
moiety or a probe. Exemplary labeling agents which are suitable for use
in the context of the these embodiments include, but are not limited to,
a fluorescent agent, a radioactive agent, a magnetic agent, a
chromophore, a bioluminescent agent, a chemiluminescent agent, a
phosphorescent agent and a heavy metal cluster.

[0194] The phrase "radioactive agent" describes a substance (i.e.
radionuclide or radioisotope) which loses energy (decays) by emitting
ionizing particles and radiation. When the substance decays, its presence
can be determined by detecting the radiation emitted by it. For these
purposes, a particularly useful type of radioactive decay is positron
emission. Exemplary radioactive agents include 99mTc, 18F,
131I and 125I.

[0195] The term "magnetic agent" describes a substance which is attracted
to an externally applied magnetic field. These substances are commonly
used as contrast media in order to improve the visibility of internal
body structures in Magnetic Resonance Imaging (MRI). The most commonly
used compounds for contrast enhancement are gadolinium-based. MRI
contrast agents alter the relaxation times of tissues and body cavities
where they are present, which, depending on the image weighting, can give
a higher or lower signal.

[0196] As used herein, the term "chromophore" describes a chemical moiety
that, when attached to another molecule, renders the latter colored and
thus visible when various spectrophotometric measurements are applied.

[0197] The term "bioluminescent agent" describes a substance which emits
light by a biochemical process

[0198] The term "chemiluminescent agent" describes a substance which emits
light as the result of a chemical reaction.

[0199] The phrase "fluorescent agent" refers to a compound that emits
light at a specific wavelength during exposure to radiation from an
external source.

[0200] The phrase "phosphorescent agent" refers to a compound emitting
light without appreciable heat or external excitation as by slow
oxidation of phosphorous.

[0201] A heavy metal cluster can be for example a cluster of gold atoms
used, for example, for labeling in electron microscopy techniques.

[0202] In some embodiments, the labeling agent is Fluorescein
isothiocyanate.

[0203] As demonstrated in the Examples section that follows, a fluorescent
agent being Fluorescein isothiocyanate (FITC) has been conjugated to HPMA
copolymeric backbone having TNP-470 and alendronate attached thereto
(HPMA copolymer-ALN-TNP-470-FITC; see, FIG. 1). The fluorescent agent was
utilized for assessing the in vivo biodistribution of the conjugate (see,
FIG. 7G) as well as to study the mechanism by which the conjugate
internalize into endothelial and human osteosarcoma cells (see, FIG. 4).
These fluorescence studies showed that the conjugate is mainly
distributed to bone tissue and that the mechanism by which the conjugate
is internalized is through a lysosomotropic pathway of cellular uptake
via clathrin-coated vesicles.

[0204] In some embodiments, the conjugate has the following structure:

##STR00005##

wherein:

[0205] x is an integer having a value such that x/(x+y+w+z) multiplied by
100 is in the range of from 0.01 to 99.9, as described herein;

[0206] y is an integer having a value such that y/(x+y+w+z) multiplied by
100 is in the range of from 0.01 to 99.9, as described herein;

[0207] w is an integer having a value such that w/(x+y+w+z) multiplied by
100 is in the range of from 2.1 to 99, as described herein; and

[0208] z is an integer having a value such that z/(x+y+w+z) multiplied by
100 is in the range of from 0.01 to 99.9.

[0209] In some embodiments, z is an integer having a value such that
z/(x+y+w+z) multiplied by 100 is in the range of from 0.01 to 10, and
depends on the labeling agent utilized and the monitoring technology.

[0210] As discussed hereinabove and is further discussed in detail
hereinbelow, the conjugates described herein were successfully prepared
by devising and successfully practicing novel processes for their
preparation. Such processes, in addition to allowing obtaining a
conjugate with a high load of alendronate, further allow for obtaining
conjugates having a low polydispersity index (PDI) and small mean size
distribution.

[0211] Hence, in some embodiments, the conjugate described herein has a
polydispersity index ranging from 1 to 1.4.

[0212] The term "polydispersity index" is a measure of the distribution of
molecular mass in a given polymer sample. PDI is a value calculated as
the weight average molecular weight divided by the number average
molecular weight (Mw/Mn). It indicates the distribution of
individual molecular masses in a batch of polymers. The PDI has a value
always greater than 1, but as the polymer chains approach uniform chain
length, the PDI approaches unity (1). Polymer-based nanocarriers similar
to HPMA copolymer often exhibit inherent structural heterogeneity of the
polymers, reflected in a high PDI value, typically higher than 1.4, and
even higher than 1.6. Homogenous size distribution of polymer conjugates
may contribute to a more defined biodistribution.

[0216] Weight average molar mass (Mw) was evaluated for the
conjugates synthesized from their SEC profiles and the Polydispersity
index (PDI) was calculated according to the formula Mw/Mn.

[0217] While reducing the present invention to practice, the present
inventors have designed and successfully practiced a novel process for
preparing a HPMA co-polymer having attached thereto alendronate, TNP-470
and optionally a labeling agent (e.g., a fluorescent agent), whereby the
load of the alendronate is greater than in currently known methodologies
for attaching bisphosphonates, and optionally other targeting moieties,
to polymers.

[0218] Currently known methodologies typically include attachment of a
targeting moiety to an already prepared polymer or co-polymer, and thus,
the load of the targeting moiety in the resulting conjugate is limited
and non-controllable.

[0219] In contrast, in the methodology described herein, monomeric units
of the polymeric backbone (HPMA) to which alendronate is attached are
first prepared and then co-polymerized with HPMA monomeric, oligomeric
and/or polymeric units. Alternatively, the alendronate-containing
monomeric units are polymerized and then co-polymerized with other
monomeric, oligomeric or polymeric units of the monomer. In some
embodiments, co-polymerization is effected upon converting at least a
pre-determined portion of the HPMA monomers, oligomers or polymers to
such that terminate with a reactive group that is capable of reacting
with TNP-470 (e.g., by means of adding a spacer that terminates with the
desired reactive group), thereby functionalizing the HPMA monomers.

[0220] In some embodiments, co-polymerization is effected upon converting
at least a pre-determined portion of the HPMA monomers, oligomers or
polymers to such that terminate with a reactive group that is capable of
reacting with a labeling agent (e.g., by means of adding a spacer that
terminates with the desired reactive group).

[0221] In some embodiments, co-polymerization is effected upon converting
at least a pre-determined portion of the HPMA monomers, oligomers or
polymers to such that include a linker, as described herein, which
optionally terminates with a reactive group that is capable of reacting
with TNP-470 (e.g., by means of adding a spacer that terminates with the
desired reactive group).

[0222] Hence, according to another aspect of some embodiments of the
present invention, there is provided a process of synthesizing the
conjugates described herein, the process comprising:

[0224] (b) co-polymerizing N-(2-hydroxypropyl)methacrylamide monomeric
units, and/or a ((N-(2-hydroxypropyl)methacrylamide) oligomeric or
polymeric units with the alendronate-containing methacrylamide monomeric
units and with N-(2-hydroxypropyl)methacrylamide-derived monomeric units
terminating with a first reactive group, to thereby obtain a polymeric
backbone which comprises a plurality of methacrylamide backbone units in
which a portion of the backbone units has an alendronate attached
thereto, and another portion of the backbone units has the reactive
group, the first reactive group being capable of coupling TNP-470; and

[0225] (c) coupling the TNP-470 and the polymeric backbone via the first
reactive group, thereby obtaining the polymeric conjugate.

[0226] In some embodiments, the process further comprises, optionally
prior to the co-polymerizing in (b), coupling a labeling agent to
N-(2-hydroxypropyl)methacrylamide monomeric units, to thereby obtain
labeling agent-containing methacrylamide monomeric units; and (b) further
comprises copolymerizing the labeling agent-containing methacrylamide
monomeric units together with alendronate-containing methacrylamide
monomeric units and N-(2-hydroxypropyl)methacrylamide-derived monomeric
units terminating with a first reactive group, to thereby obtain an
alendronate-containing copolymer having the reactive group, and labeling
agent, the reactive group being capable of coupling TNP-470.

[0227] Alternatively, the co-polymerization in (b) comprises
co-polymerizing the alendronate-containing monomeric units with the
N-(2-hydroxypropyl)methacrylamide monomeric units and/or the
((N-(2-hydroxypropyl)methacrylamide) oligomeric or polymeric units and
the N-(2-hydroxypropyl)methacrylamide-derived monomeric units terminating
with a first reactive group, and further with
N-(2-hydroxypropyl)methacrylamide-derived monomeric units terminating
with a second reactive group, wherein the second reactive group is being
capable of coupling the labeling agent.

[0228] In these embodiments, the process further comprises coupling the
labeling agent to the co-polymer, via the second reactive group. Such a
coupling can be effected prior to, concomitant with, or subsequent to
coupling the TNP-470.

[0229] The copolymerization of the alendronate-containing HPMA monomers
and the other, functionalized or non-functionalized HPMA monomers can be
effected by any polymerization method known in the art, using suitable
polymerization initiators and optionally chain transfer agents. Such
suitable polymerization initiators and chain transfer agents can be
readily identified by a person skilled in the art.

[0230] As demonstrated in the Examples section that follows, the
copolymerization of alendronate containing HPMA-derived
monomers+HPMA-derived monomers terminating with a reactive group (e.g.,
ethylenediamine)+free HPMA monomers, and optionally+an HPMA-derived
monomer comprising FITC (step (b) in the process described hereinabove),
was performed via two methodologies: (1) the "classical"
thermopolymerization methodology using, as an example,
4,4'-azobis(4-cyanovaleric acid) as a polymerization initiator; and (2)
the "reversible addition-fragmentation chain transfer" (RAFT)
polymerization technique, using, as an example,
2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride as a
polymerization initiator and
S,S'-bis(α,α'-dimethyl-α''-acetic acid)
trithiocarbonate as a chain transfer agent (TTC).

[0231] Using these two synthetic approaches, it was shown that the load of
alendronate in the conjugate, as well as other parameters of the obtained
copolymer, can be finely controlled.

[0232] Using the RAFT approach further enables to perform the
copolymerization at room temperature or at temperatures as low as
30° C.

[0233] The "reversible addition-fragmentation chain transfer" (RAFT)
polymerization technique typically involves the use of thiocarbonylthio
compounds, such as dithioesters, dithiocarbamates, trithiocarbonates, and
xanthates in order to mediate the polymerization via a reversible
chain-transfer process. This allows access to polymers with low
polydispersity and high functionality.

[0234] As exemplified in the Examples section that follows, weight average
molar mass (Mw) was determined for the conjugates synthesized from
their SEC profiles and the Polydispersity index (PDI) was calculated
according to the formula Mw/Mn. It has been shown that
conjugates polymerized by RAFT were well-dispersed, exhibiting a
considerably low and narrower PDI value of about 1.2, with a mean size
distribution of 100 nm (see, FIGS. 3D-G).

[0235] Therefore, in some embodiments, the co-polymerizing is performed
via the Reversible addition-fragmentation chain transfer (RAFT)
technique.

[0236] In some embodiments, the process is performed such that the
conjugate has a polydispersity index ranging from 1 to 1.4.

[0237] In some embodiments, the process is performed such that the
conjugate has a mean size distribution lower than 150 nm.

[0238] Generally, the TNP-470 or alendronate can be attached to the
monomeric units that form the polymeric backbone, or to the backbone
units of the copolymer, by means of a functional group that is already
present in the native molecule and/or the monomeric units of the polymer,
or otherwise can be introduced or generated by well-known procedures in
synthetic organic chemistry without altering the activity of the agent.
For example, in the case of alendronate, a terminal carboxylic group can
be generated within a monomeric HPMA, in order to form an amide with the
amine functional group of alendronate. A carboxylic functional group can
be generated, for example, by oxidizing the hydroxy group of the HPMA.
Alternatively, a carboxylic functional group (reactive group) is
generated by attaching to an HPMA monomer a spacer or a linker that
terminates with a carboxylic group.

[0239] Similarly, an alkylhalide can be generated within the HPMA
polymeric backbone or within HPMA monomers, in order to readily couple
TNP-470. Such an alkylhalide can be generated by means of a spacer and/or
linker, as described herein.

[0240] HPMA monomeric, oligomeric and polymeric units that have been
modified so as to generate a reactive group are therefore referred to
herein as HPMA-derived units or as methacrylamide units terminating by a
reactive group.

[0241] Accordingly, in some embodiments, the process further comprises
introduction of a linker to at least some of the HPMA monomeric,
oligomeric or polymeric units participating in the co-polymerization.

[0242] In some embodiments, introducing the linker is performed subsequent
to the co-polymerization.

[0243] Similarly, in some embodiments, the process further comprises
introduction of a spacer to at least some of the HPMA monomeric,
oligomeric or polymeric units participating in the co-polymerization.

[0244] In some embodiments, introducing the spacer is performed subsequent
to the co-polymerization.

[0245] In some embodiments, a plurality of functionalized HPMA monomeric
units is first prepared. The functionalized HPMA monomers include: HPMA
monomers that include a spacer and/or a linker for attaching alendronate;
HPMA monomers that include a spacer and/or a linker for attaching
TNP-470; and optionally HPMA monomers that include a spacer for attaching
a labeling agent. These functionalized HPMA monomeric units are referred
to herein as HPMA-derived units.

[0246] Then, alendronate-containing HPMA-derived monomers are prepared,
and are co-polymerized with the other functionalized HPMA monomers,
optionally in the presence of non-modified HPMA monomers (which form
"free" backbone units upon co-polymerization).

[0247] In some embodiments, alendronate-containing methacrylamide
monomeric are prepared by first preparing
N-methacryloylglycylglycylprolylnorleucine units (SEQ ID NO:7), and
thereafter conjugating thereto the alendronate, so as to obtain
N-methacryloylglycylglycylprolylnorleucyl-alendronate monomeric units
(SEQ ID NO:8).

[0248] In some embodiments, HPMA-derived methacrylamide units that
terminate with a first reactive group include
N-methacryloylglycylglycylprolylnorleucine units (SEQ ID NO:7).

[0249] In some embodiments, HPMA-derived methacrylamide units that
terminate with a second reactive group include N-methacryloylglycylglycyl
units (SEQ ID NO:9).

[0250] Since attaching alendronate, a labeling agent, a spacer and/or a
linker to HPMA units, or otherwise generating a reactive group within the
HPMA unit, involves reaction with the 2-hydroxypropyl group in these
units, such functionalized HPMA units are also referred to herein as
methacrylamide units that contain the above-described moieties or groups.

[0251] Co-polymerization is effected as described hereinabove.

[0252] Then, TNP-470 is coupled to the formed co-polymer.

[0253] The order of steps can be modified, as long as alendronate is
attached to monomeric HPMA units, prior to co-polymerization of such
units, in order to assure a high and controllable load of alendronate.

[0254] Herein, the phrases "functional group" and "reactive group" are
used interchangeably. Accordingly, "functionalized" monomers (monomeric
units), oligomers, etc. describe such monomers, oligomers, etc. that have
a reactive group, as defined and described herein.

[0255] The present inventors have utilized some of the methodologies
described herein for introducing another targeting moiety into a polymer
conjugate that further comprises a therapeutically active agent such as
an anti-angiogenesis agent.

[0256] Thus, the present inventors have further designed and successfully
prepared and practiced novel conjugates of a polymer (e.g., a
N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer), an anti-angiogenesis
agent (e.g., TNP-470) and a bone targeting agent being an oilgoaspartate
(e.g. D-Asp8 (SEQ ID NO: 1)).

[0257] As demonstrated in the Examples section that follows, an HPMA
copolymer having TNP-470 and D-Asp8 (SEQ ID NO: 1) attached thereto
has been prepared (HPMA copolymer-D-Asp8-TNP-470; see, FIG. 8). The
anti-angiogenesis activity of the conjugate has been demonstrated by the
ability to inhibit the proliferation of HUVEC by the conjugate (see, FIG.
10) and inhibition of vascular endothelial growth factor (VEGF)-induced
HUVEC migration (FIG. 11).

[0258] These results suggest that the use of these conjugates for treating
bone and bone related disorders (such as cancer and disorders
characterized by angiogenesis), is beneficial.

[0259] Therefore, according to another aspect of some embodiments of the
present invention there is provided a polymeric conjugate comprising a
polymeric backbone having attached thereto an anti-angiogenesis agent and
a bone targeting moiety, the bone targeting moiety being an oligopeptide
of aspartic acid which comprises from 2 to 100 aspartic acid residues.

[0260] The term "anti-angiogenesis agent" is as defined hereinabove.

[0261] The phrase "bone targeting moiety" describes a compound having the
capability of preferentially accumulating in hard tissues (i.e. bone
tissues) rather than any other organ or tissue, after administration in
vivo.

[0262] Oligopeptides of aspartic acid such as D-aspartate octapeptide
(D-Asp8) (SEQ ID NO: 1) have been known to accumulate in bone. These
oligopeptides bind to Hydroxyapitate (HA), the major constituent of the
bone, thereby being suitable for serving as a bone targeting moiety.

[0264] In some embodiments the oligopeptide of aspartic acid comprises
from 2 to 20 aspartic acid residues. In some embodiments the oligopeptide
of aspartic acid comprises 8 aspartic acid residues (Asp8). In some
embodiments the oligopeptide of aspartic acid consists of 8 aspartic acid
residues (Asp8, SEQ ID NO:10).

[0265] The oligopeptide can further include other amino acid residues, as
long as it includes one or more amino acid sequences that consist of two
or more aspartic acid residues. In some embodiments, such amino acid
sequences consist of 2 to 20 aspartic acid residues or of 8 aspartic acid
residues.

[0266] The aspartic acid can be D-aspartic acid and/or L-aspartic acid. In
some embodiments, the aspartic acid is D-aspartic acid.

[0270] As used herein, the term "COX-2 inhibitor" refers to a
non-steroidal drug that relatively inhibits the enzyme COX-2 in
preference to COX-1. Preferred examples of COX-2 inhibitors include, but
are no limited to, celecoxib, parecoxib, rofecoxib, valdecoxib,
meloxicam, and etoricoxib.

[0271] In some embodiments, the polymeric conjugates described herein are
composed of a polymeric backbone, formed from a plurality of backbone
units that are covalently linked to one another, wherein at least a
portion of this plurality of backbone units has an anti-angiogenesis
agent, as described herein, attached thereto, and at least another
portion of the plurality of backbone units has the bone targeting moiety
(the oligoaspartate, as described herein), attached thereto.

[0272] Those backbone units that have the anti-angiogenesis agent attached
thereto and those backbone units that have the oligoaspartate attached
thereto can be randomly dispersed within the polymeric backbone.

[0273] The polymeric backbone can further include non-functionalized
backbone units, as discussed hereinbelow, to which none of the
anti-angiogenesis agent and the oligoaspartate are attached.

[0274] In some embodiments, the polymeric backbone of the conjugates
described constitutes polymers (or co-polymers) to which the
anti-angiogenesis agent and the bone targeting moiety are attached.

[0275] Polymers which are suitable for use in the context of the present
embodiments are preferably biocompatible, non-immunogenic and non-toxic.
The polymers serve as carriers that enable specific delivery into tumor
tissue, possible due to the EPR effect described hereinabove.

[0276] As used herein, the term "polymer" describes an organic substance
composed of a plurality of repeating structural units (backbone units)
covalently connected to one another. The term "polymer" as used herein
encompasses organic and inorganic polymers and further encompasses one or
more of a homopolymer, a copolymer or a mixture thereof (a blend). The
term "homopolymer" as used herein describes a polymer that is made up of
one type of monomeric units and hence is composed of homogenic backbone
units. The term "copolymer" as used herein describes a polymer that is
made up of more than one type of monomeric units and hence is composed of
heterogenic backbone units. The heterogenic backbone units can differ
from one another by the pendant groups thereof.

[0277] The polymer is comprised of backbone units formed by polymerizing
the corresponding monomeric units whereby the anti-angiogenesis agent and
the bone targeting moiety are attached to at least a portion of the
backbone units. Some or all of these backbone units are typically
functionalized prior to conjugation so as to have a reactive group for
attaching the anti-angiogenesis agent and the bone targeting moiety.
Those backbone units that are not functionalized and/or do not
participate in the conjugations of the anti-angiogenesis agent and bone
targeting moiety are referred to herein as "free" backbone units.

[0278] The polymer may be a biostable polymer, a biodegradable polymer or
a combination thereof. The term "biostable", as used in this context of
embodiments of the invention, describes a compound or a polymer that
remains intact under physiological conditions (e.g., is not degraded in
vivo).

[0279] The term "biodegradable" describes a substance which can decompose
under physiological and/or environmental conditions into breakdown
products. Such physiological and/or environmental conditions include, for
example, hydrolysis (decomposition via hydrolytic cleavage), enzymatic
catalysis (enzymatic degradation), and mechanical interactions. This term
typically refers to substances that decompose under these conditions such
that 50 weight percents of the substance decompose within a time period
shorter than one year.

[0280] The term "biodegradable" as used in the context of embodiments of
the present invention, also encompasses the term "bioresorbable", which
describes a substance that decomposes under physiological conditions to
break down products that undergo bioresorption into the host-organism,
namely, become metabolites of the biochemical systems of the
host-organism.

[0281] The polymers can be water-soluble or water-insoluble. In some
embodiments, the polymers are water soluble at room temperature.

[0282] The polymers can further be charged polymers or non-charged
polymers. Charged polymers can be cationic polymers, having positively
charged groups and a positive net charge at a physiological pH; or
anionic polymers, having negatively charged groups and a negative net
charge at a physiological pH. Non-charged polymers can have positively
charged and negatively charged group with a neutral net charge at
physiological pH, or can be non-charged.

[0283] In some embodiments, the polymer has an average molecular weight in
the range of 100 Da to 800 kDa. In some embodiments, the polymer has an
average molecular weight lower than 60 kDa. In some embodiments, the
polymer's average molecular weight ranges from 10 kDa to 40 kDa.

[0284] Polymeric substances that have a molecular weight higher than 10
kDa typically exhibit an EPR effect, as described herein, while polymeric
substances that have a molecular weight of 100 kDa and higher have
relatively long half-lives in plasma and an inefficient renal clearance.
Accordingly, a molecular weight of a polymeric conjugate can be
determined while considering the half-life in plasma, the renal
clearance, and the accumulation in the tumor of the conjugate.

[0285] The molecular weight of the polymer can be controlled, at least to
some extent, by the degree of polymerization (or co-polymerization).

[0286] The polymer used in the context of these embodiments of the
invention can be a synthetic polymer or a naturally-occurring polymer. In
some embodiments, the polymer is a synthetic polymer.

[0287] The polymeric backbone of the polymer described herein may be
derived from, for example, polyacrylates, polyvinyls, polyamides,
polyurethanes, polyimines, polysaccharides, polypeptides,
polycarboxylates, and mixtures thereof.

[0288] Exemplary polymers which are suitable for use in the context of the
present embodiments include, but are not limited to the group consisting
of dextran, a water soluble polyamino acid, a polyethylenglycol (PEG), a
polyglutamic acid (PGA), a polylactic acid (PLA), a
polylactic-co-glycolic acid, (PLGA), a poly(D,L-lactide-co-glycolide)
(PLA/PLGA), a poly(hydroxyalkylmethacrylamide), a polyglycerol, a
polyamidoamine (PAMAM), and a polyethylenimine (PEI).

[0289] These polymers can be of any molecular weight, as described herein.

[0290] In some embodiments, the polymeric backbone is derived from a
poly(hydroxyalkylmethacrylamide) or a copolymer thereof. Such a polymeric
backbone comprises methacrylamide backbone units having attached thereto
either 2-hydroxypropyl groups or such 2-hydroxypropyl groups that have
been modified by attaching thereto (directly or indirectly) the moieties
described herein (the oligoaspartate and the anti-angiogenesis agent).

[0291] It is to be understood that the polymers as discussed herein
describe those polymers that are formed from homogenic or heterogenic,
non-functionalized monomeric units, and that the polymeric backbone
constituting the polymeric conjugate corresponds to such polymers by
being comprised of the same monomeric units, while some of these
monomeric units are functionalized, as described herein. Thus, the
polymeric backbone of the polymeric conjugate is similar to that of the
polymers described herein, and differs from the polymers by having the
above-described agents attached to some of the backbone units therein.

[0292] In each of the conjugates described herein, the bone targeting
moiety and the anti-angiogenesis agent can each be linked to the
respective portion of the backbone units in the polymeric backbone
directly, or indirectly, through a linker moiety (also referred to herein
as a linker, a linker group or a linking group), whereby, in some
embodiments, the direct/indirect linkage is designed as being cleavable
at conditions characterizing the desired bodily site (e.g., by certain
enzymes or pH), as detailed hereinbelow.

[0293] Hence, according to some embodiments of the invention, at least one
of the anti-angiogenesis agent and the bone targeting moiety is attached
to the polymer via a linker. In some embodiments, each of the
anti-angiogenesis agent and the bone targeting moiety is attached to the
polymer via a linker. The linker linking the anti-angiogenesis agent to
the polymer and the linker linking the bone targeting moiety to the
polymer may be the same or different.

[0294] The linker described herein refers to a chemical moiety that serves
to couple the anti-angiogenesis agent and/or the bone targeting moiety to
the polymer while not adversely affecting either the targeting function
of the bone targeting moiety or the therapeutic effect of the
anti-angiogenesis agent.

[0295] The linker characteristics have been described elaborately
hereinabove.

[0296] In some embodiments, each of the anti-angiogenesis agent and the
bone targeting moiety is attached to the polymer via a linker. In such a
case, the linker linking the anti-angiogenesis agent and the linker
linking the bone targeting moiety may be the same or different.

[0297] In some embodiments, only the anti-angiogenesis agent is linked to
the polymer via a biodegradable linker, thereby being released from the
polymer at the desired bodily site.

[0298] As demonstrated in the Examples section that follows, a HPMA
copolymer of TNP-470 and D-Asp8 (SEQ ID NO: 1) has been synthesized
using the biodegradable linker -[Gly-Gly-Pro-Nle]- (SEQ ID NO: 3) in
order to link TNP-470 to the polymer.

[0299] As discussed hereinabove, an exemplary linker is a Cathepsin K
cleavable linker.

[0300] Hence, in some embodiments, the linker is an
enzymatically-cleavable linker. In some embodiments the
enzymatically-cleavable linker is cleaved by an enzyme which is expressed
in tumor tissues. In some embodiments the enzymatically-cleavable linker
is cleaved by an enzyme which is overexpressed in tumor tissues.

[0301] Exemplary enzymes which are suitable for use in the context of the
present embodiments include, but are not limited to Cathepsin B,
Cathepsin K, Cathepsin D, Cathepsin H, Cathepsin L, legumain, MMP-2 and
MMP-9.

[0302] As discussed hereinabove, Cathepsin K is expressed predominantly in
osteoclasts. Therefore, in some embodiments the enzymatically-cleavable
linker is cleaved by Cathepsin K.

[0303] In some embodiments the biodegradable linker comprises an
oligopeptide having from 2 to 10 amino acid residues.

[0304] As discussed hereinabove, a Cathepsin K cleavable linker being
-[Gly-Gly-Pro-Nle]- (SEQ ID NO: 3) has been used by the present inventors
in order to conjugate TNP-470 to a HPMA polymer (see FIG. 8).
Accordingly, in some embodiments, the linker comprises
-[Gly-Gly-Pro-Nle]- (SEQ ID NO: 3).

[0305] In some embodiments, the bone targeting moiety described in the
context of these embodiments of the invention is attached to the
polymeric backbone via a biostable linker.

[0306] In some embodiments, the bone targeting moieties attached to the
polymeric backbone via an enzymatically cleavable linker, for example, a
Cathepsin K cleavable linker as described hereinabove.

[0307] In some cases, the conjugate described herein comprise an
additional spacer moiety which enables a more efficient and simpler
attachment of the anti-angiogenesis agent and/or the bone targeting
moiety to the polymeric backbone. The spacer may be further utilized in
order to attach a labeling agent to the conjugate. Such a spacer has been
described extensively hereinabove.

[0308] As illustrated in the example section that follows hereinbelow,
spacers comprising a --[NH--(CH2)2--NH]-- group and a
1-aminohaxanoyl have been used, intradispersly, in order to conjugate
TNP-470 through a -[Gly-Gly-Pro-Nle]- (SEQ ID NO: 3) Cathepsin K
cleavable linker to the HPMA copolymer (see, FIG. 8). Spacers derived
from 1-aminohexaonoic acid were used intradispersly in order to attach
D-Asp8 (SEQ ID NO: 1) and a labeling agent being FITC to the polymer
(see, FIG. 8).

[0309] In some embodiments, the conjugate may further comprise a labeling
agent, as defined herein. Such a labeling agent is described elaborately
hereinabove. In some embodiments, the labeling agent is attached to the
conjugate via a spacer, as described herein. As demonstrated in the
Examples section that follows, a labeling agent being Fluorescein
isothiocyanate (FITC) has been conjugated to a HPMA
copolymer-D-Asp8-TNP-470 via the spacer used to couple the
D-Asp8 (the D-Asp8 having an ID SEQ NO: 1) (see, FIG. 8).

[0310] The degree of loading of the anti-angiogenesis agent and the bone
targeting moiety may be expressed as mol %, as defined herein.

[0311] Thus, for example, a 1 mol % load of a bone targeting moiety
describes a polymeric conjugate composed of 100 backbone units, whereby 1
backbone unit has a targeting moiety attached thereto and the other 99
backbone units are either free or have other agents attached thereto.

[0312] The optimal degree of loading of the anti-angiogenesis agent and
bone targeting moiety for a given conjugate and a given use is determined
empirically based on the desired properties of the conjugate (e.g., water
solubility, therapeutic efficacy, pharmacokinetic properties, toxicity
and dosage requirements), and optionally on the amount of the conjugated
moiety that can be attached to a polymeric backbone in a synthetic
pathway of choice.

[0313] The % loading can be measured by methods well known by those
skilled in the art, some of which are described hereinbelow under the
Materials and Methods of the Examples section that follows.

[0314] In some embodiments, the loading of the anti-angiogenesis agent in
the polymer is greater than 1 mol %.

[0315] In some embodiments, the loading of the anti-angiogenesis agent in
the conjugate ranges from 1 mol % to 99 mol %, from 1 mol % to 50 mol %,
from 1 mol % to 20 mol %, from 1 mol % to 10 mol %, or from 1 mol % to 5
mol %.

[0316] In some embodiments the loading of the bone targeting moiety in the
polymer is greater than 1 mol %.

[0317] In some embodiments, the loading of the bone targeting moiety in
the conjugate ranges from 1 mol % to 99 mol %, from 1 mol % to 50 mol %,
from 1 mol % to 20 mol %, from 1 mol % to 10 mol %, or from 1 mol % to 5
mol %.

[0318] The number of backbone units within the polymeric backbone that
have an anti-angiogenesis agent conjugated thereto is defined herein as
"a", the number of backbone units within the polymeric backbone that have
a bone targeting moiety conjugated thereto is herein defined as "b" and
the number of free backbone units in the polymeric backbone (which are
not bound to an additional moiety) is herein defined as "d".

[0319] Accordingly, in some embodiments, the conjugate described herein
can be represented by the general formula I:

[A1]d[A2-L1-B]a[A3-L2-D]b Formula I

[0320] wherein:

[0321] a is an integer having a value such that x/(x+y+w) multiplied by
100 is in the range of from 0.01 to 99.9;

[0322] b is an integer having a value such that y/(x+y+w) multiplied by
100 is in the range of from 0.01 to 99.9; and

[0323] d is an integer having a value such that w/(x+y+w) multiplied by
100 is in the range of from 0.01 to 99.9;

[0324] A1, A2 and A3 are each backbone units covalently
linked to one another and forming the polymeric backbone, wherein:

[0325] B is the anti-angiogenesis agent as defined hereinabove;

[0326] D is the bone targeting moiety as defined hereinabove; and

[0327] each of the L1 and L2 is independently a linker as
defined hereinabove;

[0328] such that [A2-L1-B] is a backbone unit having attached
thereto the anti-angiogenesis agent; and

[0329] [A3-L2-B] is a backbone unit having attached thereto the
bone targeting moiety;

[0330] wherein each of the [A1], the [A2-L1-B] and the
[A3-L2-D] is either a terminal backbone unit being linked to
one of the [A1], the [A2-L1-B] and the
[A3-L2-D], or is linked to at least two of the [A1], the
[A2-L1-B] and the [A3-L2-B] and the A1, A2
and/or A3 are linked to one another to thereby form the polymeric
backbone.

[0331] In embodiments where the polymeric conjugate is derived from HPMA,
A1 is a hydroxypropylmethacrylamide unit; and A2 and A3 is
a methacrylamide unit, as discussed hereinabove.

[0332] In some embodiments, the conjugate described herein can be
represented by the general formula IIa:

##STR00006##

[0333] wherein a, b and d are as defined herein.

[0334] In some embodiments the conjugate has the following structure:

##STR00007##

[0335] wherein a and b are each independently an integer having a value
such that a/(a+b+d) multiplied by 100 and/or b/(a+b+d)×100 are in
the range of from 0.01 to 15; and d is an integer having a value such
that d/(a+b+d) multiplied by 100 is in the range of from 70 to 99.9.

[0336] It would be appreciated that a, b and d can be controlled as
desired by selecting the mol ratio of the respective monomeric units used
for forming the polymeric conjugate.

[0337] According to another aspect of some embodiments of the present
invention, there is provided a process for preparing the conjugates
described herein. The process, according to these embodiments, is
effected by:

[0338] (a) co-polymerizing a plurality of monomeric units of said
polymeric backbone, wherein a portion of said plurality comprises
monomeric units terminating by a first reactive group, and another
portion of said plurality comprises monomeric units terminating by a
second reactive group, to thereby obtain a co-polymer comprising a
polymeric backbone that comprises a plurality of backbone units, wherein
a portion of said backbone units has said first reactive group and
another portion of said backbone units has said second reactive group,
said first reactive group being capable of reacting with said
anti-angiogenesis agent and said second reactive being capable of
reacting with said bone targeting moiety;

[0339] (b) coupling the bone targeting moiety to the co-polymer via the
first reactive group, thereby obtaining a bone targeting
moiety-containing copolymer; and

[0340] (c) coupling the anti-angiogenesis agent to the co-polymer via the
second reactive group; thereby obtaining the conjugate.

[0341] The phrase "oligomeric units of the polymer", or simply "oligomeric
units", as used herein throughout, describes a polymer comprised of 2-50
backbone units. Hence, the polymeric backbone of the conjugate described
herein may be constructed by copolymerizing the functionalized monomeric
units, as described hereinabove, together with non-functionalized
monomeric or oligomeric units that compose the backbone.

[0342] In some embodiments, each of the functionalized monomeric units can
first be polymerized, so as to form a functionalized oligomer bearing a
plurality of the first reactive groups and a functionalized oligomer
bearing a plurality of the second reactive groups, and these oligomers
can be co-polymerized with each other and with the non-functionalized
oligomeric or monomeric units.

[0343] In some embodiments, only one of the functionalized monomeric units
is polymerized so as to form a functionalized oligomer, which is then
co-polymerized with the other functionalized monomeric units and
non-functionalized monomeric or oligomeric units.

[0344] As used herein throughout, the term "functionalized" describes a
monomer or an oligomer that terminates with one or more reactive groups.

[0345] As used herein throughout, a "reactive group" describes a chemical
group that is capable of reacting with another group so as to form a
chemical bond, typically a covalent bond. Optionally, an ionic or
coordinative bond is formed.

[0346] A reactive group is termed as such if being chemically compatible
with a reactive group of an agent or moiety that should be desirably
attached thereto. For example, a carboxylic group is a reactive group
suitable for conjugating an agent or a moiety that terminates with an
amine group, and vice versa.

[0347] A reactive group can be inherently present in the monomeric units,
oligomeric units and/or bone targeting moiety and the anti-angiogenesis
agent, or be generated therewithin by terms of chemical modifications of
the chemical groups thereon or by means of attaching to these chemical
groups a spacer or a linker that terminates with the desired reactive
group, as described herein.

[0348] Co-polymerizing the monomers or oligomers described herein can be
effected by any of the polymerization methods known in the art, using
suitable polymerization initiators or any other catalysts known in the
art.

[0349] As discussed hereinabove, it has been shown that co-polymerization
via the Reversible addition-fragmentation chain transfer (RAFT) technique
yields conjugates having a low polydispersity index and small mean size
distribution.

[0350] Therefore, in some embodiments, the co-polymerization is performed
via the reversible addition-fragmentation chain transfer (RAFT)
technique, as exemplified in the Examples section that follows.

[0351] In some embodiments, the anti-angiogenesis agent is conjugated to
the polymer prior to the conjugation of the bone targeting moiety. In
some embodiments, the bone targeting moiety is coupled to the polymer
prior to conjugating the anti-angiogenesis agent.

[0352] Each of the first and the second reactive groups can be protected
prior to the respective conjugation thereto. In such cases, the process
further comprises deprotecting each of the reactive groups prior to the
respective conjugation.

[0353] This allows a controlled conjugation of, for example, the
anti-angiogenesis agent to those backbone units that comprises a
biodegradable linker.

[0354] It should be appreciated that the monomeric units, spacers and
linkers utilized for coupling the anti-angiogenesis agent and/or the bone
targeting moiety to the polymer are designed so as to allow a smooth and
efficient conjugation of the respective moiety and an optimal performance
of the obtained conjugate, as discussed elaborately hereinabove.

[0355] Thus, in some embodiments, the process is further effected by
preparing the monomeric units or oligomeric units that comprise the first
and second reactive groups.

[0356] In some embodiments, monomeric units having attached thereto a
spacer terminating with a protected first reactive group are prepared.
Exemplary such monomeric units are methacrylamide units (derived from
HPMA, as defined herein) having attached thereto a protected Gly-Gly
group (SEQ ID NO:11).

[0357] Similarly, monomeric units having attached thereto a linker and
optionally a spacer, terminating with a protected second reactive group
are prepared.

[0358] The ratio between the above-described monomeric units and
non-functionalized monomeric or oligomeric units that form a part of the
formed polymer determines, at least in part, the mol ratio of the
respective bone targeting moiety and anti-angiogenesis moiety in the
formed conjugate.

[0359] Co-polymerizing the monomeric and/or oligomeric units bearing the
reactive groups results in a functionalized polymer, bearing the first
and second reactive groups (optionally protected with respective
protecting groups).

[0360] In some embodiments, the bone targeting moiety and/or the
anti-angiogenesis moiety are modified prior to being conjugated to the
functionalized polymer, so as to include reactive groups that are
compatible with the first and second reactive groups, respectively, of
the functionalized polymer.

[0361] Such a modification can be effected by means of attaching a spacer
and/or a linker to the bone targeting moiety and/or the anti-angiogenesis
agent prior to the conjugation thereof to the functionalized polymer.

[0362] Hence, in some embodiments, the process is further effected by
preparing such modified bone targeting moiety and/or anti-angiogenesis
agent.

[0363] The linkers and/or spacers interposed between the polymeric
backbone and the moieties conjugated thereto are designed so as to
exhibit the properties described elaborately hereinabove with respect
thereto.

[0364] Similarly, embodiments of the process described herein also apply
for other processes described herein (e.g., for preparing a HPMA
copolymer-alendronate-TNP-470 conjugate as described herein).

[0365] The spacer may be varied in length and in composition depending on
steric consideration and may be used to space the angiogenesis agent
and/or bone targeting moiety form the polymer, thereby enabling easier
synthesis of the conjugate and/or improved performance of the formed
conjugate, as detailed hereinabove.

[0366] In some embodiments the process further comprises attaching a
labeling agent, as defined herein, to the formed conjugate. The labeling
agent can be attached to either of functionalized monomeric units, prior
to co-polymerization or to the formed co-polymer.

[0367] In some embodiments, the labeling agent is attached to the
co-polymer concomitantly with the bone targeting moiety. Alternatively,
it is attached prior to or subsequent to attaching the bone targeting
moiety and/or the anti-angiogenesis agent.

[0368] In some embodiments, the process comprises co-polymerizing, along
with the functionalized and non-functionalized monomeric or oligomeric
units described herein, monomeric units terminating with a third reactive
group, the third reactive group being for conjugating thereto a labeling
agent or any other additional moiety, as described herein.

[0369] Thus, each of the conjugates described in any of the embodiments of
the invention, may further include an additional moiety conjugated
thereto. Such an additional moiety can be conjugated either to monomeric
units within and throughout the polymeric backbone, or be attached at one
or both ends of the polymeric backbone.

[0370] Such an additional moiety can be a labeling agent, as described
herein, or an additional targeting moiety or an additional
therapeutically active agent, which may improve the performance of the
formed conjugate. Such an additional moiety can further be a moiety that
improves the solubility, bioavailability, and/or any other desired
feature of the formed conjugate.

[0371] The conjugates described hereinabove may be prepared, administered
or otherwise utilized in any of the aspects of embodiments of the
invention, either as is, or as a pharmaceutically acceptable salt,
enantiomers, diastereomers, solvates, hydrates or a prodrug thereof.

[0372] The phrase "pharmaceutically acceptable salt" refers to a charged
species of the parent compound and its counter ion, which is typically
used to modify the solubility characteristics of the parent compound
and/or to reduce any significant irritation to an organism by the parent
compound, while not abrogating the biological activity and properties of
the administered compound. The neutral forms of the compounds are
preferably regenerated by contacting the salt with a base or acid and
isolating the parent compound in a conventional manner. The parent form
of the compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents, but otherwise the salts
are equivalent to the parent form of the compound for the purposes of the
present invention.

[0373] The phrase "pharmaceutically acceptable salts" is meant to
encompass salts of the moieties and/or conjugates which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When conjugates
according to embodiments of the invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting the
neutral (i.e., non-ionized) form of such conjugates with a sufficient
amount of the desired base, either neat or in a suitable inert solvent.
Examples of pharmaceutically acceptable base addition salts include
sodium, potassium, calcium, ammonium, organic amino, or magnesium salt,
or a similar salt. When conjugates according to embodiments of the
invention contain relatively basic functionalities, acid addition salts
can be obtained by contacting the neutral form of such conjugates with a
sufficient amount of the desired acid, either neat or in a suitable inert
solvent. Examples of pharmaceutically acceptable acid addition salts
include, but are not limited to, those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as
well as the salts derived from relatively nontoxic organic acids like
acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,
suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,
p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also
included are salts of amino acids such as arginate and the like, and
salts of organic acids like glucuronic or galactunoric acids and the like
(see, for example, Berge et al., "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific conjugates of
the present invention contain both basic and acidic functionalities that
allow the conjugates to be converted into either base or acid addition
salts.

[0374] The neutral forms of the conjugates are preferably regenerated by
contacting the salt with a base or acid and isolating the parent
conjugate in a conventional manner. The parent form of the conjugate
differs from the various salt forms in certain physical properties, such
as solubility in polar solvents, but otherwise the salts are equivalent
to the parent form of the conjugate for the purposes of the present
invention.

[0375] In an example, a pharmaceutically acceptable salt of alendronate is
utilized. An exemplary such salt is sodium alendronate. An
alendronate-containing conjugate can therefore comprise a sodium salt of
alendronate.

[0376] In another example, a pharmaceutically acceptable salt of aspartate
is utilized.

[0377] The term "prodrug" refers to an agent, which is converted into the
active compound (the active parent drug) in vivo. Prodrugs are typically
useful for facilitating the administration of the parent drug. The
prodrug may also have improved solubility as compared with the parent
drug in pharmaceutical compositions. Prodrugs are also often used to
achieve a sustained release of the active compound in vivo.

[0378] The conjugates described herein may possess asymmetric carbon atoms
(optical centers) or double bonds; the racemates, diastereomers,
geometric isomers and individual isomers are encompassed within the scope
of embodiments of the invention.

[0379] As used herein, the term "enantiomer" describes a stereoisomer of a
compound that is superposable with respect to its counterpart only by a
complete inversion/reflection (mirror image) of each other. Enantiomers
are said to have "handedness" since they refer to each other like the
right and left hand. Enantiomers have identical chemical and physical
properties except when present in an environment which by itself has
handedness, such as all living systems.

[0380] The conjugates described herein can exist in unsolvated forms as
well as solvated forms, including hydrated forms. In general, the
solvated forms are equivalent to unsolvated forms and are encompassed
within the scope of the present invention.

[0381] The term "solvate" refers to a complex of variable stoichiometry
(e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a
solute (the conjugate described herein) and a solvent, whereby the
solvent does not interfere with the biological activity of the solute.
Suitable solvents include, for example, ethanol, acetic acid and the
like.

[0382] The term "hydrate" refers to a solvate, as defined hereinabove,
where the solvent is water.

[0383] Certain conjugates of the present invention may exist in multiple
crystalline or amorphous forms. In general, all physical forms are
equivalent for the uses contemplated by the present invention and are
intended to be within the scope of the present invention.

[0384] As discussed hereinabove, the conjugates described herein (also
referred to herein throughout as polymeric conjugates) comprise a bone
targeting moiety being either alendronate or an oligopeptide of
aspartate, which enables the targeting of the conjugate to bone and bone
related structures. Due to the anti-angiogenesis/anti-proliferative
activity exhibited by the moieties attached to the polymer and the formed
conjugate as a whole, each of the conjugates described herein can be
beneficially used for treating bone and bone related disease and
disorders.

[0385] Hence, according to another aspect of some embodiments of the
present invention there are provided methods of treating a bone related
disease or disorder in a subject in need thereof. These methods are
effected by administering to the subject a therapeutically effective
amount of any of the conjugates described herein.

[0386] Accordingly, according to another aspect of some embodiments of the
present invention there are provided uses of any of the conjugates
described herein as a medicament. In some embodiments, the medicament is
for treating a bone-related disease or disorder.

[0387] According to another aspect of some embodiments of the present
invention, the conjugates described herein are identified for use in the
treatment of a bone related disease or disorder.

[0388] As used herein, the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including, but
not limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means, techniques and
procedures by practitioners of the chemical, pharmacological, biological,
biochemical and medical arts.

[0389] The phrase "a bone related disease or disorder" describes a disease
or disorder wherein bone formation, deposition, or resorption is
abnormal, especially those characterized by excessive angiogenesis. The
phrase "bone related disease or disorder" also encompasses disease and
disorders occurring in bodily sites other than bone which evolved from a
bone related disease or disorder such as, for example, metastasis of bone
cancer in another organ and diseases and disorders which evolved in other
bodily sites and affect bone tissues.

[0391] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical symptoms of
a condition or substantially preventing the appearance of clinical or
aesthetical symptoms of a condition. When the treatable disease is bone
cancer, this term encompasses any inhibition of tumor growth or
metastasis, or any attempt to inhibit, slow or abrogate tumor growth or
metastasis.

[0392] It is noted herein that by targeting an anti-angiogenesis agent via
the methodologies described herein, the toxicity of the anti-angiogenesis
agent is substantially reduced, due to the conjugate selectivity towards
bone tissues. Consequently, besides the use of the conjugates described
herein in a clinically evident disease, optionally in combination with
other drugs, these conjugates may potentially be used as a long
term-prophylactic for individuals who are at risk for relapse due to
residual dormant cancers. The use of non-toxic targeted conjugates for
the treatment of asymptomatic individuals who are at risk for relapse of
osteosarcoma, as an example, may lead to a major paradigm shift in cancer
treatment from current methods where treatment is generally not initiated
until a bone related disease such as osteosarcoma becomes clinically
evident.

[0393] The term "subject" (alternatively referred to herein as "patient")
as used herein refers to an animal, preferably a mammal, most preferably
a human, who has been the object of treatment, observation or experiment.

[0394] As demonstrated in the Examples section that follows and discussed
elaborately hereinabove, it has been shown that the conjugates described
herein inhibit angiogenesis as well as cell proliferation and therefore
can be utilized for the treatment of bone related disease and disorders
characterized by pathologically excessive angiogenesis wherein the
inhibition of angiogenesis and/or cell proliferation is beneficial.

[0395] Hence, in some embodiments the bone related disease or disorder is
associated with angiogenesis.

[0396] Tumor growth and metastasis are particularly dependent on the
degree of angiogenesis. Tumor angiogenesis is the proliferation of a
network of blood vessels that penetrate into cancerous tumors in order to
supply nutrients and oxygen and remove waste products, thus leading to
tumor growth. Tumor angiogenesis involves hormonal stimulation and
activation of oncogenes, expression of angiogenic growth factors,
extravasation of plasma protein, deposition of a provisional
extracellular matrix (ECM), degradation of ECM, and migration,
proliferation and elongation of endothelial capillaries. Inhibition of
further vascular expansion has therefore been the focus of active
research for cancer therapy.

[0397] Hence, in some embodiments, the bone related disease or disorder is
selected from the group consisting of bone cancer metastases and bone
cancer.

[0398] The terms "cancer" and "tumor" are used interchangeably herein to
describe a class of diseases in which a group of cells display
uncontrolled growth (division beyond the normal limits). The term
"cancer" encompasses malignant and benign tumors as well as disease
conditions evolving from primary or secondary tumors. The term "malignant
tumor" describes a tumor which is not self-limited in its growth, is
capable of invading into adjacent tissues, and may be capable of
spreading to distant tissues (metastasizing). The term "benign tumor"
describes a tumor which is not-malignant (i.e. does not grow in an
unlimited, aggressive manner, does not invade surrounding tissues, and
does not metastasize). The term "primary tumor" describes a tumor that is
at the original site where it first arose. The term "secondary tumor"
describes a tumor that has spread from its original (primary) site of
growth to another site, close to or distant from the primary site.

[0399] The term "bone cancer" describes tumors that arise from the tissues
of the bone. The term "bone cancer", as used herein, further encompasses
tumors in tissues located in proximity to bone structures and associated
with bone such as cartilage, bone cavity and bone marrow. The term "bone
cancer" further encompasses cancer which evolved from bone cells (i.e.
primary tumor), as well as cancer cells which have "breaken away",
"leaked", or "spilled" from a primary tumor located in bone, entered the
lymphatic and/or blood vessels, circulated through the lymphatic system
and/or bloodstream, settled down and proliferated within normal tissues
elsewhere in the body thereby creating a secondary tumor. For example,
metastases originating from osteosarcoma can be frequently found in the
lungs and in other organs. These lesions produce an osteoid and therefore
can be targeted with bone targeting moieties, as described herein.

[0400] Bone cancer is found most often in the bones of the arms and legs,
but it can occur in any bone.

[0401] Bone cancers are also known as sarcomas. There are several types of
sarcomas of bone, depending upon the kind of bone tissue where the tumor
developed. Exemplary types of bone cancers that are treatable according
to embodiments of the invention include, but are not limited to,
osteosarcoma, Ewing's sarcoma, chondrosarcoma, fibrosarcoma, malignant
giant cell tumor, and chordoma.

[0402] Osteosarcoma is the most common type of primary bone cancer and
classified as a malignant mesenchymal neoplasm in which the tumor
directly produces defective osteoid (immature bone). It is a highly
vascular and extremely destructive malignancy that most commonly arises
in the metaphyseal ends of long bones. Several strategies were proposed,
such as immune-based therapy, tumor-suppressor or suicide gene therapy,
or anticancer drugs that are not commonly used in osteosarcoma [Quan et
al. Cancer Metastasis Rev 2006; 10: 707-713]. However, still one-third of
patients die from this devastating cancer, and for those with
unresectable disease there are no curative systemic therapies.

[0403] The term "bone metastases" describes cancer evolving form a primary
tumor located in bodily site other than bone but metastasizing to the
bone (i.e. a secondary tumor). Cancers that commonly metastasize, or
spread, to the bones include breast cancer, lung cancer, thyroid cancer,
prostate cancer, some brain cancers and cancers of the kidney.

[0404] For example, prostate cancer is the most common cancer of males in
industrialized countries and the second leading cause of male cancer
mortality. Prostate cancer predominantly metastasizes to bone, but other
organ sites are affected including the lung, liver, and adrenal gland.
Bone metastases incidence in patients with advanced metastatic disease is
approximately 70%. Bone metastases are associated with considerable
skeletal morbidity, including severe bone pain, pathologic fracture,
spinal cord or nerve root compressions, and hypercalcemia of malignancy.

[0405] As discussed hereinabove, the conjugates described herein may be
further utilized for monitoring bone related disease or disorders. In
such a case the conjugate further comprises a labeling agent, as defined
herein, for easy detection of the conjugate in the body of the patient,
using well known imaging techniques. For example, in the case of the bone
related disease or disorder being bone cancer the detection of the
conjugate, as assessed by the level of labeling agent signal, can serve
to detect bone cancer metastases in bodily sites other than bone.

[0406] Hence, according to another aspect of some embodiments of the
invention, there are provided methods of monitoring a bone related
disease or disorder in a subject. The method according to these
embodiments of the invention is effected by administering to the subject
any of the conjugates described herein, having a labeling agent attached
to the polymer, as described herein, and employing an imaging technique
for monitoring a distribution of the conjugate within the body or a
portion thereof.

[0407] Accordingly, according to another aspect of some embodiments of the
present invention there are provided uses of any of the conjugates
described herein, having a labeling agent as described herein, as
diagnostic agents and/or in the manufacture of a diagnostic agent for
monitoring a bone related disease or disorder.

[0408] According to another aspect of some embodiments of the present
invention, each of the conjugates described herein, which comprises a
labeling agent, is identified for use as a diagnostic agent, for
monitoring a bone related disease or disorder.

[0409] Suitable imaging techniques include, but are not limited to,
positron emission tomography (PET), gamma-scintigraphy, magnetic
resonance imaging (MRI), functional magnetic resonance imaging (FMRI),
magnetoencephalography (MEG), single photon emission computerized
tomography (SPECT) computed axial tomography (CAT) scans, ultrasound,
fluoroscopy and conventional X-ray imaging. The choice of an appropriate
imaging technique depends on the nature of the labeling agent, and is
within the skill in the art. For example, if the labeling agent comprises
Gd ions, then the appropriate imaging technique is MRI; if the labeling
agent comprises radionuclides, an appropriate imaging technique is
gamma-scintigraphy; if the labeling agent comprises an ultrasound agent,
ultrasound is the appropriate imaging technique, etc.

[0410] According to another aspect of the present invention there is
provided a pharmaceutical composition comprising, as an active
ingredient, any of the conjugates described herein and a pharmaceutically
acceptable carrier

[0411] Accordingly, in any of the methods and uses described herein, any
of the conjugates described herein can be provided to an individual
either per se, or as part of a pharmaceutical composition where it is
mixed with a pharmaceutically acceptable carrier.

[0412] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the conjugates described herein (as active
ingredient), or physiologically acceptable salts or prodrugs thereof,
with other chemical components including but not limited to,
physiologically suitable carriers, excipients, lubricants, buffering
agents, antibacterial agents, bulking agents (e.g. mannitol),
antioxidants (e.g., ascorbic acid or sodium bisulfite), anti-inflammatory
agents, anti-viral agents, chemotherapeutic agents, anti-histamines and
the like. The purpose of a pharmaceutical composition is to facilitate
administration of a compound to a subject. The term "active ingredient"
refers to a compound, which is accountable for a biological effect.

[0413] The terms "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be interchangeably used
refer to a carrier or a diluent that does not cause significant
irritation to an organism and does not abrogate the biological activity
and properties of the administered compound.

[0414] Herein the term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of a
drug. Examples, without limitation, of excipients include calcium
carbonate, calcium phosphate, various sugars and types of starch,
cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0415] Techniques for formulation and administration of drugs may be found
in "Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton,
Pa., latest edition, which is incorporated herein by reference.

[0416] Pharmaceutical compositions for use in accordance with the present
invention thus may be formulated in conventional manner using one or more
pharmaceutically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the compounds into
preparations which can be used pharmaceutically. Proper formulation is
dependent upon the route of administration chosen. The dosage may vary
depending upon the dosage form employed and the route of administration
utilized. The exact formulation, route of administration and dosage can
be chosen by the individual physician in view of the patient's condition
(see e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p. 1).

[0417] The pharmaceutical composition may be formulated for administration
in either one or more of routes depending on whether local or systemic
treatment or administration is of choice, and on the area to be treated.
Administration may be done orally, by inhalation, or parenterally, for
example by intravenous drip or intraperitoneal, subcutaneous,
intramuscular or intravenous injection, or topically (including
ophtalmically, vaginally, rectally, intranasally).

[0418] Formulations for topical administration may include but are not
limited to lotions, ointments, gels, creams, suppositories, drops,
liquids, sprays and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be necessary
or desirable.

[0419] Compositions for oral administration include powders or granules,
suspensions or solutions in water or non-aqueous media, sachets, pills,
caplets, capsules or tablets. Thickeners, diluents, flavorings,
dispersing aids, emulsifiers or binders may be desirable.

[0420] Formulations for parenteral administration may include, but are not
limited to, sterile solutions which may also contain buffers, diluents
and other suitable additives. Slow release compositions are envisaged for
treatment.

[0421] The amount of a composition to be administered will, of course, be
dependent on the subject being treated, the severity of the affliction,
the manner of administration, the judgment of the prescribing physician,
etc.

[0422] The pharmaceutical composition may further comprise additional
pharmaceutically active or inactive agents such as, but not limited to,
an anti-bacterial agent, an antioxidant, a buffering agent, a bulking
agent, a surfactant, an anti-inflammatory agent, an anti-viral agent, a
chemotherapeutic agent and an anti-histamine.

[0423] According to some embodiments, the pharmaceutical composition
described herein is packaged in a packaging material and identified in
print, in or on the packaging material, for use in the treatment of a
bone related disease or disorder.

[0424] According to other embodiment of the present invention, the
pharmaceutical composition is packaged in a packaging material and
identified in print, in or on the packaging material, for use in
monitoring a bone related disease or disorder.

[0425] Compositions of the present invention may, if desired, be presented
in a pack or dispenser device, such as an FDA approved kit, which may
contain one or more unit dosage forms containing the active ingredient.
The pack may, for example, comprise metal or plastic foil, such as a
blister pack. The pack or dispenser device may be accompanied by
instructions for administration. The pack or dispenser may also be
accommodated by a notice associated with the container in a form
prescribed by a governmental agency regulating the manufacture, use or
sale of pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling approved by
the U.S. Food and Drug Administration for prescription drugs or of an
approved product insert.

[0426] In any of the methods, uses and compositions described herein, the
conjugates described herein can be utilized in combination with
additional therapeutically active agents. Such additional agents include,
as non-limiting examples, chemotherapeutic agents, anti-angiogensis
agents, hormones, growth factors, antibiotics, anti-microbial agents,
anti-depressants, immunostimulants, and any other agent that may enhance
the therapeutic effect of the conjugate and/or the well-being of the
treated subject.

[0429] The term "consisting essentially of" means that the composition,
method or structure may include additional ingredients, steps and/or
parts, but only if the additional ingredients, steps and/or parts do not
materially alter the basic and novel characteristics of the claimed
composition, method or structure.

[0430] As used herein, the singular form "a", "an" and "the" include
plural references unless the context clearly dictates otherwise; For
example, the term "a compound" or "at least one compound" may include a
plurality of compounds, including mixtures thereof.

[0431] Throughout this application, various embodiments of this invention
may be presented in a range format. It should be understood that the
description in range format is merely for convenience and brevity and
should not be construed as an inflexible limitation on the scope of the
invention. Accordingly, the description of a range should be considered
to have specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example, description
of a range such as from 1 to 6 should be considered to have specifically
disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2
to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within
that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of
the breadth of the range.

[0432] Whenever a numerical range is indicated herein, it is meant to
include any cited numeral (fractional or integral) within the indicated
range. The phrases "ranging/ranges between" a first indicate number and a
second indicate number and "ranging/ranges from" a first indicate number
"to" a second indicate number are used herein interchangeably and are
meant to include the first and second indicated numbers and all the
fractional and integral numerals therebetween.

[0433] It is appreciated that certain features of the invention, which
are, for clarity, described in the context of separate embodiments, may
also be provided in combination in a single embodiment. Conversely,
various features of the invention, which are, for brevity, described in
the context of a single embodiment, may also be provided separately or in
any suitable subcombination or as suitable in any other described
embodiment of the invention. Certain features described in the context of
various embodiments are not to be considered essential features of those
embodiments, unless the embodiment is inoperative without those elements.

[0434] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below find
experimental support in the following examples.

EXAMPLES

[0435] Reference is now made to the following examples, which together
with the above descriptions, illustrate the invention in a non limiting
fashion.

Materials and Methods

[0436] Materials:

[0437] All reactions requiring anhydrous conditions were performed under
argon or nitrogen atmosphere.

[0438] Chemicals and solvents were either A.R. grade or purified by
standard techniques.

[0451] All other chemical reagents, including salts and solvents, were
purchased from Sigma-Aldrich.

[0452] All animal procedures were performed in compliance with Tel Aviv
University, Sackler School of Medicine guidelines and protocols approved
by the Institutional Animal Care and Use Committee. Mice's body weight
and tumor size were measured three times a week.

[0458] HUVEC were plated at 10,000 cells/well onto 24-well culture plates
in EBM-2 supplemented with 5% FBS and incubated for 24 hours (37°
C.; 5% CO2). Medium was replaced with 2.5% endothelial cell basal
medium-2 (EBM-2) supplemented with 1% Endothelial cell growth supplement
(ECGS). Human osteosarcoma Saos-2 or MG-63-Ras cells were plated at 2500
cells/well in DMEM supplemented with 5 FBS and incubated for 24 hours
(37° C.; 5% CO2). The medium was then replaced with DMEM
supplemented with 10% FBS. Cells were exposed to ALN, TNP-470, and HPMA
copolymer-ALN-TNP-470 conjugate or with equivalent concentrations of
combinations of free ALN and TNP-470 at serial dilutions. HUVEC were also
incubated with or without 1 μM of cathepsin-K inhibitor III. Control
cells were grown in the presence or absence of growth factors. HUVEC or
Saos-2 viable cells were counted by a Z1 Coulter® Particle Counter
(Beckman Coulter®) or by XTT reagent respectively after 72 hours of
incubation.

[0459] Isobolograms of ALN and TNP-470 Drug Combination Treatments:

[0460] IC50 represents the concentration of a drug that is required
for 50% inhibition in vitro. The IC30, 50, 70 values of treatment
with ALN, TNP-470 and their respective combinations from HUVEC
proliferation assay were collected. IC30, 50, 70 values of TNP-470
and ALN were marked on X, Y axis respectively and a line which represents
additive effect was drawn between each inhibitory concentration (IC). The
combination index (CI) of each treatment was calculated according to the
classic isobologram equation
CI=[(D)1/(Dx)1]+[(D)2/(Dx)2] as previously described
[Chou T. C. Pharmocol Rev 2006; 58:621-681]. Area on the right side of
each IC additive line represents antagonist effect while the left side
represents synergic effect.

[0461] Characterization of HPMA-ALN-TNP-470 Conjugate:

[0462] (a) Determination of ALN Content:

[0463] The formation of chromophoric complex between ALN and Fe3+
ions in perchloric acid solution was used to spectrophotometrically
determine the ALN content by spectrophotometry. Briefly, 0.1 ml conjugate
(concentration 2-10 mgram/ml) was mixed with 0.1 ml 4 mM FeCl3 and
0.8 ml 0.2 M HClO4 and absorbance at 300 nm was measured against
blank. The calibration curve was prepared by using an ALN solution at a
concentration range of 0-3 mM.

[0464] (b) Estimation of TNP-470 Content:

[0465] The content of TNP-470 was estimated from the content of NH2
groups in the HPMA copolymer-ALN-NH2 precursor (shown in FIG. 1,
middle structure), assuming that the TNP-470 binding was quantitative.
The content of NH2 groups was determined by ninhydrin method using
an amine containing monomer (N-(3-aminopropyl)methacrylamide) as the
calibration sample (modified from Duncan, at el. J Control Release 2001;
74: 135-146).

[0471] The mean hydrodynamic diameter of the conjugate was evaluated using
a real time particle analyzer (NANOSIGHT LM20®) containing a
solid-state, single mode laser diode (<20 mW, 655 nm) configured to
launch a finely focused beam through a 500 μl sample chamber. HPMA
copolymer-ALN-TNP-470 conjugate was dissolved in phosphate buffered
saline (PBS) to final concentrations of 0.5, 1 and 2 mg/ml. The samples
were then injected into the chamber by syringe and allowed to equilibrate
to unit temperature (23° C.) for 30 seconds. The particles
dynamics were visualized at 30 frames per second (fps) for 60 seconds at
640×480 resolution by a coupled charge device (CCD) camera. The
paths the particles take under Brownian motion over time were analyzed
using Nanoparticle Tracking Analysis (NTA) software. The diffusion
coefficient and hence sphere equivalent hydrodynamic radius of each
particle was separately determined and the particle size distribution
profiles were generated. Each sample was measured three times in
triplicates, and the results represent the mean diameter.

[0472] Hydroxyapatite Binding Assay:

[0473] In order to assess the ability of HPMA copolymer-ALN-TNP-470
conjugate to bind to bone mineral, its binding potency to hydroxyapatite
(HA) was evaluated. HPMA copolymer-ALN-TNP-470 conjugate was dissolved in
phosphate buffered saline (PBS), pH 7.4 (1 mg/ml). The conjugate solution
(500 μl) was incubated with hydroxyapatite powder (15 mg), in 500
μl PBS, pH 7.4. HPMA copolymer-Gly-Phe-Leu-Gly was used as control.
Incubated samples were centrifuged at 6000 RPM for 3 minutes and a sample
from the upper layer (100 μl) was collected at selected time points.
FPLC analysis using HighTrap desalting column (Amersham®) was used
for detection of unbound conjugate in the samples (FPLC conditions:
AKTA® Purifier®, mobile phase 100% DDW, 2 ml/minute, 215 nm).
Hydroxyapatite binding kinetic analysis of the conjugate was performed
using the Unicorn® AKTA® software. Areas under the curve (AUC)
were calculated from chromatographs at each time point. AUC of each
hydroxyapatite incubated conjugate chromatogram was normalized to percent
AUC of conjugate sample in the absence of hydroxyapatite used as control.

[0475] For all experiments, human umbilical vain endothelial cells (HUVEC)
and Saos-2 human osteosarcoma cells were seeded on sterile 13 mm cover
glasses in 35 mm culture dishes 24 hours before incubation with
fluorescein isothiocyanate (FITC) labeled HPMA copolymer-ALN-TNP-470
conjugate and left to reach 90% confluence. HUVEC and Saos-2 cells were
then incubated with 10 μM FITC-HPMA copolymer ALN-TNP-470 conjugate
for 12 hours. Following incubation, cells were washed several times with
cold phosphate buffered saline (PBS), fixed with 3.5% paraformaldehyde
for 15 minutes at room temperature (RT) and washed with PBS again. For
counter staining, cells were permeabilized with 0.1% Triton-X100 for 3
minutes and rinsed with PBS again. For confocal imaging of FITC-labeled
HPMA copolymer ALN-TNP-470 conjugate cellular uptake by HUVEC, nuclei
were labeled using propidium iodide (10 μg/ml) and cover glasses were
mounted by Antifade® mounting media. Alternatively, actin filaments
were labeled using phalloidin-TRITC conjugate (50 μg/ml, 40 minutes at
RT) and cover glasses were mounted by Vectashild®DAPI containing
medium. For conjugate endosomal pathway internalization analysis, HUVEC
and Saos-2 cells were incubated with 10 μM FITC-HPMA copolymer
ALN-TNP-470 conjugate for 6 hours. Following incubation cells were washed
several times with cold PBS, starved for 45 minutes in serum free medium
at 37° C. and incubated with 40 μg/ml Alexa® Fluor 594
human transferrin for 1 hour at 37° C. Cells were then fixed and
mounted as described before. All slides were kept at 4° C. in dark
until confocal microscopy analysis was preformed.

[0476] Confocal Microscopy:

[0477] Cellular uptake, internalization and colocalization of FITC-labeled
HPMA copolymer ALN-TNP-470-FITC conjugate were monitored utilizing a
Zeiss Meta LSM 510 and a Leica TCS SP5 confocal imaging systems with
60× oil objectives. All images were taken using a multi-track
channel acquisition to prevent emission cross-talk between fluorescence
dyes. Single XY, XZ plane-images were acquired in 1024×1024
resolution. Images from Z stack acquisition were processed as separate
channels using Huygens® deconvolution software and overlaid as a
single image.

[0479] Cell migration assays were performed using modified 8 μm Boyden
chambers coated with 10 μg/ml fibronectin. HUVEC (15×104
cells/100 μl) were challenged with HPMA copolymer ALN-TNP-470
conjugate or with combinations of free ALN+free TNP-470 at equivalent
concentrations and were added to the upper chamber of the transwell for 4
hours incubation. Following incubation, cells were allowed to migrate to
the underside of the chamber for 4 hours in the presence or absence of
Vascular endothelial growth factor (VEGF) (20 ng/ml) in the lower
chamber. Cells were then fixed with ice-cold menthol and stained using
Hema 3 Stain System. The stained migrated cells were imaged using Nikon
TE2000E inverted microscope integrated with Nikon DS5 cooled CCD camera
by 10× objective, brightfield illumination. Migrated cells from the
captured images per membrane were counted using NIH image software.
Migration was normalized to percent migration, with 100% representing
VEGF dependent migration of cells which were not incubated with free or
HPMA-conjugated ALN and TNP-470.

[0480] Capillary-Like Tube Formation Assay:

[0481] The surface of 24-well plates was coated with Matrigel®
basement membrane (50 μl/well; 10 mg/ml) on ice and was allowed to
polymerize at 37° C. for 30 minutes. HUVEC (3×104
cells) were challenged with HPMA copolymer ALN-TNP-470 conjugate or with
combinations of free ALN+free TNP-470 at equivalent concentrations and
were seeded on coated plates in the presence of complete EGM-2 medium.
After 8 hours of incubation (37° C.; 5% CO2), wells were
imaged using Nikon TE2000E inverted microscope integrated with Nikon DS5
cooled CCD camera by 4× objective, brightfield illumination. Images
were analyzed for total tube area using Nikon NIS elements image
software.

[0482] Miles Vascular Permeability Assay:

[0483] Balb/c male mice were injected subcutaneously (s.c.) with TNP-470,
HPMA copolymer-ALN-TNP-470 conjugate (30 mg/kg TNP-470 equivalents) or
saline (n=5 mice/group). Three days later, a modified Miles assay was
performed as previously described [Claffey et al. 1996, Cancer Res 56:
172-181; Miles & Miles 1952, J Physiol 118:228-257]. Briefly, Evans blue
dye (100 μl of a 1% solution in 0.9% NaCl) was injected into the
retro-orbital plexus of the mice. Ten minutes later, 50 μl of human
VEGF165 (1 ng/μl) or PBS were injected intradermally into the
pre-shaved back skin. Twenty minutes later, the animals were killed, and
an area of skin that included the entire injection site was removed.
Evans blue dye was extracted from the skin by incubation with formamide
for 5 days at room temperature, and the absorbance of the extracted dye
was measured at 620 nm. Data is expressed as mean±standard error of
the mean (s.e.m.).

[0485] SCID male were inoculated s.c. with 5×105
mCherry-labeled MG-63-Ras human osteosarcoma. Mice bearing 70 mm3
tumors were injected s.c. with combination of free ALN and TNP-470 (1:1,
30 mg/kg), FITC-labeled HPMA copolymer-ALN-TNP-470 conjugate (30 mg/kg
q.o.d.×3 times TNP-470-equivalent dose) or saline (n=5 mice/group).
Therapy was initiated at a relatively early state (70 mm3) in order
to imitate a metastatic scenario as well as an early primary
osteosarcoma. Tumor progression was monitored by caliper measurement
(width×length2×0.52) and by CRI® Maestro non-invasive
intravital imaging system. At termination, tumors were dissected, weighed
and analyzed. Data is expressed as mean±standard error of the mean
(s.e.m.).

[0487] CRI Maestro® non-invasive fluorescence imaging system was used
to follow tumor progression of mice bearing mCherry-labeled MG-63-Ras
human osteosarcoma tumors and for biodistribution studies of FITC-labeled
HPMA copolymer-ALN-TNP-470 conjugate. Mice were maintained on a
non-fluorescent diet (Harlan) for the whole period of the experiment.
Mice were anesthetized using ketamine (100 mg/kg) and xylazine (12
mg/kg), treated with a depilatory cream (Veet®) and placed inside the
imaging system. Alternatively, selected organs from mice were dissected
and placed inside the imaging system. Multispectral image-cubes were
acquired through 550-800 nm spectral range in 10 nm steps using
excitation (575-605 nm) and emission (645 nm longpass) filter set. Mice
autofluorescence and undesired background signals were eliminated by
spectral analysis and linear unmixing algorithm. Additionally, dissected
tumors were fixed in 4% PFA and imaged as whole-mount by confocal
microscopy as described earlier in order to assess the conjugate
accumulation in the tumor site.

[0491] In vitro data from proliferation assays on HUVEC, MG-63-Ras and
Saso-2 cells, HUVEC's migration and capillary-like tube formation
expressed as mean±standard deviation (s.d.). In vivo data of Miles
assay and evaluation of antitumor activity of HPMA copolymer-ALN-TNP-470
conjugate was expressed as mean±standard error of the mean (s.e.m.).
Statistical significance was determined using an unpaired t-test.
P<0.05 was considered statistically significant. All statistical tests
were two-sided.

Example 1

Synthesis of HPMA Copolymer-ALN-TNP-470 Conjugate

[0492] The general synthesis of a HPMA-ALN-TNP-470 conjugate according to
some embodiments of the present invention is depicted in FIG. 1.

[0498] Synthesis of fluorescein thiourea (FITC) containing monomer
N-methacryloylaminopropyl-FITC (MA-FITC): FITC (1 gram, 2.57 mmol) and
N-(3-aminopropyl)methacrylamide hydrochloride (0.92 gram, 5.14 mmol) were
dissolved in 5 ml dimethylformamide (DMF) and the solution was cooled to
4° C. diisopropylethylamine (DIPEA) (1.79 ml, 10.3 mmol) in 2 ml
of DMF was thereafter added dropwise and the reaction mixture was stirred
at 4° C. for 2 days. The reaction mixture was then poured into 100
ml water (pH of about 4-5) and the pH was adjusted to about 4 by 6 N HCl.
The precipitate was filtered off, washed with water, and vacuum dried
over P2O5.

[0501] In a first synthetic approach, MA-Gly-Gly-Pro-Nle-ALN(SEQ ID NO:
8), MA-Gly-Gly-Pro-Nle-NH-ethylene-NH2 (SEQ ID NO: 13) (for
conjugating TNP-470), HPMA and MA-FITC (optional) are dissolved in water
in the presence of 4,4'-azobis(4-cyanovaleric acid) (VA-501) as a
co-polymerization initiator. The solution is bubbled with nitrogen for 10
minutes, the ampoule is sealed, and copolymerization is performed at
60° C.

[0502] The relative amounts of the various monomeric units can be varied
as desired, so as to determine the load of the alendronate, the TNP-470
and the PITC (if present) in the formed polymer. The reaction conditions
can further be manipulated, so as to determine the degree of
polymerization or to incorporate other moieties such as tyrosine groups
in order to radiolabel the conjugate

[0504] In another example, MA-Gly-Gly-Pro-Nle-ALN (SEQ ID NO: 8) (73 mg),
MA-Gly-Gly-Pro-Nle-NH-ethylene-NH2 (SEQ ID NO: 13) (55 mg) and HPMA
(200 mg) were dissolved in 2 ml water in the presence of
4,4'-azobis(4-cyanovaleric acid) (VA-501, 3 mg). The solution was bubbled
with nitrogen for 10 minutes, the ampoule sealed, and the mixture
polymerized at 60° C. for 24 hours.

[0505] In a second synthetic approach, a reversible addition-fragmentation
chain transfer (RAFT) polymerization technique was used.

[0506] MA-Gly-Gly-Pro-Nle-ALN (SEQ ID NO: 8),
MA-Gly-Gly-Pro-Nle-NH(CH2)2NH2 (SEQ ID NO: 13), HPMA, and
MA-FITC (optional) are dissolved in water in the presence of
2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044) as an
initiator and S,S'-bis(α,α'-dimethyl-α''-acetic acid)
trithiocarbonate as a chain transfer agent (TTC). The solution is bubbled
with nitrogen for 30 minutes, sealed in ampoule, and co-polymerization is
performed at 30° C.

[0508] Both polymers were purified by dissolving in water and
precipitating into an excess of acetone (3 times); following each
precipitation, the precipitate was washed with acetone. Finally, the
polymers were dissolved in 15 ml of water; pH adjusted to 12 with 1 N
NaOH, and dialyzed against DI water for 24 hours at 4° C. (MWCO
12-14 kDa) to remove excess ALN monomer. The sample was freeze-dried
after dialysis.

[0509] The content of ALN, FITC and amine in the HPMA conjugate was
estimated as described in the methods section hereinabove. In an
exemplary conjugate synthesized via the first synthesis approach, as
described hereinabove, the content of ALN was determined to be 0.42
mmol/gram (7.0 mol %), the content of FITC was determined to be 0.04
mmol/gram (0.6 mol %), and the content of amine (for use in the
estimation of % of bound TNP-470) was determined to be 0.24 mmol/gram
(4.3 mol %). In another exemplary conjugate synthesized via the first
synthesis approach but without FITC and while using a different
concentration of alendronate-containing monomeric units, the content of
ALN was determined to be 0.18 mmol/gram (3.2 mol %), and the content of
amine was determined to be 0.36 mmol/gram (6.4 mol %).

[0510] In an exemplary conjugate synthesized via the second synthesis
approach (RAFT), as detailed hereinabove, the content of ALN was
determined to be 7.7 mol %, the content of FITC was determined to be 0.1
mol %, and the content of amine was determined to be 6.3 mol %.

[0511] It is therefore shown that in any of synthetic approaches described
herein, the load of alendronate can be controlled as desired and further,
a relatively high load can be obtained.

[0512] Synthesis of HPMA-ALN-TNP-470 conjugate: HPMA
copolymer-ALN-NH2 (150 mg) was dissolved in 6 ml of
dimethylformamide (DMF; if necessary, a small amount of water was added
to dissolve the polymer) and the solution was cooled to 4° C.
Then, TNP-470 (150 mg) in 1 ml DMF was added. The reaction mixture was
stirred at 4° C. in the dark for 12 hours. The conjugate was
thereafter precipitated into acetone and purified by reprecipitation (3
times) from an aqueous solution into an excess of acetone. The
precipitate was washed with acetone and the residue was dissolved in
water and dialyzed for 1 day at 4° C. (MWCO 12-14 kDa) against DI
water. The product was isolated by free-drying.

[0513] The final product was purified and characterized by reverse phase
preparative HPLC. HPMA-ALN-TNP-470 conjugate eluted as a single peak with
a retention time of 20 minutes as evaluated by size exclusion
chromatography (SEC) (see, FIG. 3B).

[0514] Quantitative evaluation of HPMA-ALN-TNP-470 conjugate size
distribution: The molecular weight and polydispersity of the two
conjugates synthesized, namely, the conjugate polymerized by the
classical polymerization method (polymerization I conjugate) and the
conjugate polymerized by "living polymerization" RAFT (polymerization II
conjugate), were estimated by SEC exhibiting an apparent Mw of 80
kDa (FIGS. 3D and 3E respectively). Additionally, the hydrodynamic
diameter size distribution of the HPMA copolymer-ALN-TNP-470 conjugates
was determined using an optical analyzer. The conjugate polymerized by
the classical polymerization method had a PDI of ˜1.62 with a mean
size distribution of 241 nm whereas the conjugate polymerized by "living
polymerization" RAFT was well-dispersed exhibiting a considerably lower
and narrower PDI of ˜1.2 with a mean size distribution of 100 nm
(FIG. 3F). The values of mean size distribution of the first conjugates,
synthesized by the classical polymerization method, clearly indicate that
associates formed under the experimental conditions used.

Example 2

Effect of Combination Treatment of ALN and TNP-470 on Proliferation of
Endothelial Cells In Vitro

[0515] Prior to conjugation of ALN and TNP-470 to HPMA copolymer backbone,
the nature of the inhibitory effect of ALN and TNP-470 as a combined
therapy on endothelial cells proliferation in vitro was evaluated. HUVEC
were challenged with free or combined ALN and TNP-470. The results,
presented in FIG. 2A show that combination treatments of ALN and TNP-470
decreased the IC's of the drugs as single treatments. ALN inhibited cell
proliferation at inhibitory concentration IC30, 50, 70 of 10, 50, 90
μM, respectively, and TNP-470 inhibited cell proliferation at
IC30, 50, 70 of 0.00025, 0.1, 1000 nM, respectively. Combination
treatments I (serial concentrations of ALN and TNP-470 0.01 nM) and II
(serial concentrations of TNP-470 and ALN 10 μM) inhibited HUVEC
proliferation at IC30, 50, 70 of 0.2, 10, 30 μM and 0.00001,
0.004, 40 nM, respectively.

[0516] Combination index (CI)-isobologram equation allowed quantitative
determination of drug interactions, where CI<1, =1, or >1 indicated
synergism, additive effect, or antagonism, respectively. Next, data from
combination treatments were calculated according to CI equation and were
used to generate isobolograms at IC30, 50, 70 of HUVEC proliferation
by ALN-TNP-470 combinations. As shown in FIG. 2B, combination treatment I
had synergistic inhibitory effect on HUVEC at IC30, 50, 70 with CI
of 0.055, 0.3, and 0.89. Combination treatment II had synergistic effect
at IC50, 70 with CI of 0.23, 0.121 and additive effect at IC30
with CI of 1.025.

Example 3

Binding of a HPMA-ALN-TNP-470 Conjugate to Bone Mineral Hydroxyapatite

[0517] One of the main characteristics of ALN besides its anti-angiogenic
and antitumor activities is its pharmacokinetic profile which exhibits a
strong affinity to bone mineral under physiological conditions. To
determine if the activity of ALN is retained following
polymer-conjugation, the affinity of the conjugate to bone mineral was
evaluated by in vitro hydroxyapitate binding assay and FPLC analysis
using Hitrap desalting column. As shown in FIG. 3B, unbound conjugates
eluted as a single peak with a retention time of 1.9 minutes. AUC
decreased in correlation with hydroxyapitate incubation time. Following 2
minutes of incubation with hydroxyapitate, 50% of the HPMA copolymer
ALN-TNP-470 conjugate in the solution was bound to hydroxyapitate (see,
FIG. 3C). This rapid binding rate to hydroxyapitate decreased after 10
minutes and finally reached a plateau after 175 minutes of incubation
time with 92% of the HPMA copolymer ALN-TNP-470 conjugate bound to
hydroxyapitate.

[0518] Following chemical characterization, the ability of FITC-labeled
HPMA copolymer ALN-TNP-470 conjugate to internalize into endothelial and
human osteosarcoma cells and the mechanism by which it internalizes was
studied. HUVEC and Saos-2 osteosarcoma cells were incubated with the
conjugate, fixed, permeabilzed and stained with the nuclei marker
propidium iodide (PI). Confocal microscopy was performed by separately
multi-channel tracking for PI (red) and FITC-labeled conjugate (green).

[0519] As shown in FIG. 4A, following 12 hours of incubation, the
conjugate accumulated mostly in the cytoplasm of HUVEC and of Saos-2
cells as observed in the single plane image.

[0520] To evaluate the conjugate cellular localization and to eliminate
optical artifacts, Z-stack of 5.7 μm with 28 slices and X, Z slice was
captured and analyzed. As shown in FIGS. 4B and 4C, the conjugate was
found to be located at the same focal plane as the nuclei, confirming its
intracellular uptake.

[0521] Further examination of the conjugate cellular internalization was
preformed using phalloidin (red) for actin filaments staining and DAPI
for nuclei staining. As shown in FIGS. 4D-K, the staining revealed
accumulation of the conjugate mainly around the nuclei in HUVEC (FIGS.
4D-G) and Saos-2 cells (FIG. 4H-K).

[0522] The conjugate was capable of internalizing into HUVAC and Saos-2
cells as demonstrated by colocalization of 82% of the conjugate with
transferrin in HUVEC cells (FIGS. 4L-O) and of 71% in Saos-2 cells (FIGS.
4P-S). These high percentages of colocalization suggest a lysosomotrophic
pathway of cellular uptake via clathrin-coated vesicles.

[0523] To determine whether HPMA copolymer ALN-TNP-470 conjugate is active
in vitro and that the bound drugs retained their antitumor and
anti-angiogenic activity following polymer-conjugation, the inhibitory
effect of the conjugate on HUVEC, Saos-2 and MG-63-Ras human osteosarcoma
cell proliferation was examined.

[0524] The results, presented in FIG. 5, show that endothelial cell growth
supplement (ECGS)-induced proliferation of HUVEC was inhibited similarly
by the combination of free ALN+free TNP-470 and the HPMA
copolymer-ALN-TNP-470 conjugate at ALN and TNP-470 equivalent
concentrations, exhibiting an IC50 of 0.7 and 1 nM, and had
cytotoxic effect at doses higher than 1 and 10 nM, respectively.

[0527] HPMA alone was inert in vitro and in vivo (data not shown), in
agreement with previously published data [Duncan et al. J Control Release
2001; 74: 135-146].

[0528] In order to validate that HPMA copolymer-ALN-TNP-470 conjugate is
active mainly upon the release of ALN and TNP-470 by cathepsin K cleavage
mechanism, the inhibition of HUVEC proliferation by HPMA
copolymer-ALN-TNP-470 conjugate in the presence of cathepsin K inhibitor
III was evaluated. HPMA copolymer-ALN-TNP-470 conjugate inhibited the
proliferation of HUVEC at a 4-logs higher concentration in the presence
of cathepsin K inhibitor III than in its absence. Following 72 hours,
there was probably some free ALN and TNP-470 released hydrolytically from
HPMA copolymer-ALN-TNP-470 conjugate which led to the inhibition of
proliferation of HUVEC at concentrations higher than 4 μM
ALN-equivalent concentrations.

[0529] The effect of HPMA copolymer ALN-TNP-470 conjugate on vascular
endothelial growth factor (VEGF)-induced HUVEC migration was examined.
Migration was assessed by counting the number of cells that migrated
through the membranes towards the chemoattractant VEGF during a 4 hour
period following 4 hours of treatment with a combination of free ALN+free
TNP-470 or conjugated HPMA-ALN-TNP-470.

[0533] Both conjugates synthesized by classical and by RAFT polymerization
reactions exhibited similar effects on the in vitro assays described
hereinabove. Therefore, the narrowly dispersed and smaller in diameter
RAFT-polymerized conjugate was chosen for all in vivo studies due to an
expected improved biodistribution.

[0534] To determine whether HPMA copolymer-ALN-TNP-470 conjugate is able
to reduce microvessel hyperpermeability a modified Miles assay was used.
Evans blue dye was injected to the retro-orbital plexus and immediately
thereafter the vascular permeability-induced factor VEGF was injected
into the shaved flank of Balb/c mice. Evans blue binds to plasma proteins
and therefore extravasates along with them at sites of increased
permeability. VEGF-induced extravasation of Evans blue dye was remarkably
inhibited in mice treated with free combination of ALN and TNP-470 and
with HPMA copolymer-ALN-TNP-470 conjugate compared to vehicle treated
mice by 87% (P=0.002) and 92% (P=0.001), respectively (see, FIGS. 7A and
7B respectively).

[0536] H & E staining of tumor sections of MG-63-Ras human osteosarcoma
tumors treated with combination of free TNP-470 and ALN or with the
conjugate revealed that tumor sections from control mice consisted of
poorly differentiated tumor cells invasive through the muscle layer with
central calcified areas allowing the HA-targeting of the conjugate with
ALN. Conjugate-treated tumors were almost regressed showing cholesterol
deposits with connective tissue and giant cells and macrophages around
them as signs of regression (FIG. 6F). CD34 staining (used as an
endothelial cell marker) showed reduction of 39% (p=5.5×10-11)
in microvessel density (MVD) of combination of free ALN with TNP-470
(76±14 microvessels/mm2) and 74% (P=4.7×10-19)
reduction in MVD of conjugate-treated tumors (32±9
microvessels/mm2) vs. control group of mice (125±16
microvessels/mm2) (n=5 mice per group; FIG. 6F).

[0537] Fluorescence imaging of organs dissected from mice treated with
FITC-labeled HPMA copolymer-ALN-TNP-470 conjugate showed greater
intensity of FITC-fluorescence spectrum (composed images of unmixed
multispectral cubes) in bone tissues then in the spleen, heart, lungs,
kidneys and liver (FIG. 6G). Some fluorescence is shown in the kidneys
due to renal excretion of the conjugate.

Example 10

HPMA Copolymer-TNP-470 Conjugates with Octa-D-Aspartate as a Bone
Targeting Moiety

[0538] Two HPMA copolymer-TNP-470 conjugates with octa-D-aspartate as a
bone targeting moiety are described herein (polymers denoted as HP1 and
HP2). The samples containing aspartate were prepared using different
polymer precursors, obtained either by "thermo" polymerization (AIBN as
the initiator; HP1) or "photo" polymerization (HP2). The molecular weight
profiles of these two polymers are different.

[0542] MA-Gly-Gly-OH (SEQ ID NO: 15) (10 grams, 0.05 mol) and
2,4,5-trichlorophenol (12 grams, 0.06 mol) were dissolved in 100 ml DMF
and cooled to (-5)-(-10)° C. Dicyclohexylcarbodiimide (DCC; 12.5
grams, 0.06 mol) in 50 ml DMF was added slowly, and the reaction mixture
was stirred for 3 hours at (-5) to (-10)° C., overnight at
4° C., and 4 hours at room temperature. Acetic acid (about 0.5 ml)
was then added and stirring continued for 1 hour. The DCU
(dicyclohexylurea) was removed by filtration and the filtrate was slowly
poured into about 800 ml of cooled water. The precipitate was filtered
and dried under vacuum at room temperature. The product was purified by
recrystallization from ethanol (dissolved in about 200 ml of boiling
ethanol, then left to cool to room temperature, and placed into a
refrigerator for crystallization). The product (crystals) was filtered,
washed with a small amount of ethanol, and dried under vacuum thereby
yielding 15 grams of the desired Compound 1 (79%).

[0545] MA-Gly-Gly-Pro-Nle-ethylenediamine (SEQ ID NO: 13) (100 mg) and
diisopropylethylamine (DIPEA; 77 μl) were dissolved in 2 ml of THF and
cooled to 0° C. Fmoc-Cl (115 mg) in 3 ml of THF was added slowly
under stirring. The reaction mixture was stirred at room temperature for
an additional 1 hour. Then the solvent was removed under vacuum, and the
residue was purified by chromatography using a silica gel column with
elution solvents: CHCl3, followed by CHCl3/MeOH 9/1 yielding
the desired Compound 2.

[0549] HPMA (200 mg), MA-Gly-Gly-Pro-Nle-ethylenediamine-Fmoc (Compound 2;
77 mg; SEQ D NO:17), MA-Gly-Gly-OTcp (SEQ ID NO: 16; Compound 1; 44 mg),
and 3 mg of AIBN were dissolved in 2.5 ml of DMSO and the solution was
bubbled with N2 for 10 minutes. The polymerization ampoule was
sealed and polymerization proceeded at 50° C. for 24 hours. The
polymer was precipitated into an excess of acetone and purified by
dissolving in methanol and precipitating into acetone 3 times. The final
product was dried under vacuum, yielding 260 mg of the desired Compound
4.

[0550] HPMA-OTcp-NH-Fmoc was also synthesized by photo-polymerization, as
follows: 2,2-Dimethoxy-2-phenylacetophenone (DMPAP; 40 mM), instead of
AIBN, was used as the initiator. The polymerization was conducted under
light (about 1000 lm/m2) for 24 hours at room temperature.
Purification was performed as described hereinabove for
thermo-polymerization.

[0553] Deprotection of Fmoc and Binding of TNP-470 to the HPMA-D-Asp8
[P-FD8-TNP470 (Compound 6)]:

[0554] HPMA-FD8-NH-Fmoc (Compound 5; 150 mg) was dissolved in DMF (5 ml)
and piperidine (1.2 ml) was added. The reaction mixture was stirred for 1
hour at room temperature. Then, the reaction mixture was acidified with
acetic acid and precipitated into acetone (the polymer did not
precipitate into acetone without acidifying). The copolymer was purified
by washing 3 times with CHCl3 to remove the piperidine-acetic acid
salt, re-precipitated 3 times from methanol into acetone, and dried under
vacuum. The polymer was further purified by FPLC (acetate buffer, pH
5.5). The polymer fraction was concentrated and pH adjusted to about 8 by
NaHCO3, followed by dialysis against DI water for 24 hours at
4° C. (MWCO 6-8 kDa) to remove the salt. The sample was
freeze-dried after dialysis. HPMA-FD8-NH2 (100 mg) was dissolved in
DMF (3 ml) and cooled to 4° C. Then TNP-470 (100 mg) in 1 ml of
DMF was added. The reaction mixture was stirred at 4° C. in dark
for 12 hours. The conjugate was thereafter precipitated into acetone, and
purified by dissolving in water and precipitating in acetone for 3 times;
after each precipitation the precipitate was washed with acetone. The
conjugate was further purified by FPLC (acetate buffer, pH 5.5), dialyzed
for 1 day at 4° C. (MWCO 6-8 kDa) and freeze-dried yielding the
desired HPMA-D-Asp8-TNP-470 conjugate (Compound 6).

Effect of HP1 and HP2 Conjugates on Growth Inhibition and Migration of
HUVEC

[0559] As an attempt to evaluate whether. TNP-470, when bound to HPMA
copolymer, retained its antiangiogenic effect, proliferation and
migration assays were performed.

[0560] The proliferation of HUVEC was inhibited similarly by free TNP-470
and conjugated TNP-470 (see, FIG. 10). The effect of HP1 and HP2
conjugates on vascular endothelial growth factor (VEGF)-induced HUVEC
migration was examined. Migration was assessed by counting the number of
cells that migrated through the membranes towards the chemoattractant
VEGF during a 4 hour period following 4 hours of treatment with free
TNP-470 or conjugated TNP-470.

[0561] As shown in FIG. 11, treatments with free or conjugated TNP-470 at
equivalent concentrations of 0.1, 1 and 10 nM dramatically inhibited the
chemotactic migration response to VEGF (see, FIG. 11A for HP1 and FIG.
11B for HP2). HUVEC basal migration in the absence of VEGF was 33%
compared to VEGF induced cells.

[0564] Although the invention has been described in conjunction with
specific embodiments thereof, it is evident that many alternatives,
modifications and variations will be apparent to those skilled in the
art. Accordingly, it is intended to embrace all such alternatives,
modifications and variations that fall within the spirit and broad scope
of the appended claims.

[0565] All publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by reference into
the specification, to the same extent as if each individual publication,
patent or patent application was specifically and individually indicated
to be incorporated herein by reference. In addition, citation or
identification of any reference in this application shall not be
construed as an admission that such reference is available as prior art
to the present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.